ADVANCES IN ATOMIC SPECTROSCOPY
Volume 7
9 2002
Volumes 1-5"
Published by JAI PRESSINC.
Volume 6:
Special Issue of MicrochemicalJournal Published by Elsevier Science B.V.
Volume 7:
Published by Elsevier Science B.V.
ADVANCES IN ATOMIC SPECTROSCOPY
Editor: JOSEPH SNEDDON Department of Chemistry McNeese State University Lake Charles, Louisiana VOLUME 7
9 2002
2002 Elsevier A m s t e r d a m - B o s t o n - L o n d o n - N e w Y o r k - O x f o r d - Paris San D i e g o -
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Table of Contents Preface ................................................................................................ xiii Contents a n d Contributors to Volumes 1-6 in the series ............................... xv Short B i o g r a p h y o f Contributors to Volume 7 .............................................. xix A b s t r a c t o f Chapters in Volume 7 .............................................................. xxvii
Chapter 1: ~
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Use of atomic spectrometry (ICP-MS) in the clinical laboratory .................................................................................... 1 Introduction ................................................................................ 1 Atomic spectrometry techniques in the clinical laboratory ................................................................................... 3 2.1 Requirements for trace element analysis in the clinical laboratory ........................................................... 3 2.2 Flame atomic spectrometry ............................................. 6 2.3 Electrothermal atomization atomic absorption Spectrometry ................................................................... 7 2.4 Inductively coupled plasma optical emission Spectrometry ................................................................... 8 2.5 Inductively coupled plasma mass spectrometry ........... 11 Inductively coupled plasma mass spectrometry ...................... 11 3.1 Fundamentals and recent development of the technique 11 3.2 Techniques for sample introduction .............................. 13 Determination of trace element concentrations in body fluids and tissues ................................................................................ 15 4.1 Background ................................................................... 15 4.2 Sample preparation ....................................................... 15 4.3 Interferences and their control ...................................... 16 4.3.1 Spectral interferences .......................................... 16 4.3.2 Non-spectral interferences .................................. 18 4.4 Applications .................................................................. 18 Stable isotopes tracers: a tool for research and diagnosis ....... 20 5.1 Background ................................................................... 20 5.2 Biological and analytical constraints for human studies using stable isotopes as tracers ..................................... 22 5.3 Determination of stable isotopes ratios fro tracer studies in humans by ICP-MS ................................................... 23 5.3.1 Copper and nickel ............................................... 24 5.3.2 Calcium ............................................................... 25 5.3.3 Iron ...................................................................... 26
5.3.4 Selenium ............................................................. 27 5.4 Other applications of isotope measurements ................ 28 6. Speciation ................................................................................ 29 6.1 Background ................................................................... 29 7. Reference methods and reference materials for trace element analysis .................................................................................... 30 References ............................................................................... 30 C h a p t e r 2: New developments in hydride generation-atomic spectrometry ............................................................................. 53 1. Introduction ............................................................................. 53 2. Novel hydride generation ........................................................ 54 2.1 Electrochemical hydride generation ............................. 54 2.2 HG utilizing fast gas-liquid separation ......................... 60 2.3 HG with immobilized borohydride on ion-exchange column and moveable reduction bed ............................ 62 2.4 Vesicle-assisted hydride generation .............................. 64 3. Advances of methods of atomization ...................................... 68 3.1 Atomization interferences in the gas phase .................. 68 3.2 In-situ trapping HG/electrothermal atomic absorption spectrometry .................................................................. 71 4. Chemical interferences in liquid phase and pre-reduction ...... 74 5. Hyphenated techniques ............................................................ 80 5.1 HPLC/on-line treatment/HG/atomic spectrometry ....... 80 5.2 CE/HG/ICP-AES (or ICP-MS) ..................................... 84 6. Applications ............................................................................. 87 6.1 Arsenic .......................................................................... 87 6.2 Selenium ........................................................................ 92 6.3 Antimony and bismuth .................................................. 99 6.4 Germanium, tin and lead ............................................. 102 6.5 Miscellaneous .............................................................. 103 7. Conclusion ............................................................................. 103 References ............................................................................. 104 C h a p t e r 3: Analysis of biological materials by double focusing-inductively coupled plasma-mass spectrometry (DF-ICP-MS) ............... 117 1. Introduction ........................................................................... 117 2. Instrumentation ...................................................................... 121 2.1 Magnetic and electrostatic mass analysers ................. 121 2.1.1. Magnetic mass analysers .................................. 121 2.1.2. Electrostatic analysers ...................................... 123
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Double focusing: forward and reverse Nier-Johnson geometries ................................................................... 124 2.3 Performance of commercial D F - I C P - M S instruments 125 2.4 Peak shapes and sensitivity ......................................... 127 2.5 Data collection ............................................................ 127 Elemental analysis of biological samples ............................. 129 3.1 Spectral interferences .................................................. 129 3.1.1 Blood, plasma and serum samples ................... 129 3.1.2 Urine samples ................................................... 133 3.1.3 Tissue samples .................................................. 134 3.1.4 Arsenic and selenium ........................................ 135 3.1.5 Noble metals ..................................................... 135 3.1.6 Rare earth elements, scandium and yttrium ..... 136 3.2 Matrix interferences .................................................... 137 3.2.1 Serum and urine samples .................................. 137 3.2.2 The case of selenium ......................................... 138 3.3 Sensitivity and limits of detection .... 139 3.4 Biomedical applications .............................................. 141 3.5 Applications of food samples ...................................... 143 3.6 Application to environmental biological samples ...... 148 3.7 Determination of radionuclides in biological s a m p l e s . 149 Isotope ratio measurements ................................................... 150 4.1 Accuracy of isotope ratios by D F - I C P - M S ................. 150 4.1.1 Mass bias ........................................................... 150 4.1.2 Detector dead time ............................................ 151 4.1.3 Blanks ............................................................... 152 4.1.4 Isobaric interferences ........................................ 153 4.2 Precision of isotope ratio m e a s u r e m e n t s .................... 153 4.3 Resolution of spectral interferences ............................ 155 4.4 Tracer studies ............................................................... 156 4.5 Paleoanthropological applications .............................. 157 4.6 Isotope dilution analysis ............................................. 157 Trace metal speciation ........................................................... 158 5.1 High performance liquid chromatography ( H P L C ) .... 159 5.1.1 Size exclusion ................................................... 159 5.1.2 Ion exchange ..................................................... 161 5.1.3 Selenium speciation .......................................... 161 5.1.4 D N A adducts quantification ............................. 165 5.1.5 Organic solvents-induced interferences ........... 167 5.2 Gas chromatography ................................................... 167
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5.3 Capillary electrophoresis (CE) .................................... 169 5.4 Off-line strategies ........................................................ 170 5.5 Future of D F - I C P - M D S for speciation ....................... 171 References ............................................................................. 172 C h a p t e r 4: Field-flow fractionation-inductively coupled plasma-mass spectrometry .......................................................................... 179 1. Introduction ........................................................................... 179 2. General overview .................................................................. 182 2.1 F F F modes ................................................................... 186 2.2 F F F sub-techniques ..................................................... 187 2.2.1 Sedimentation F F F (SdFFF) ............................. 188 2.2.2 Thermal F F F (ThFFF) ...................................... 190 2.2.3 Electrical F F F (E1FFF) ..................................... 192 2.2.4 Flow F F F (FIFFF) ............................................. 194 2.3 Instrumentation and optimization ............................... 198 2.3.1 Instrumentation ................................................. 198 2.3.2 Optimization ..................................................... 199 2.4 Quantitative analysis by F F F ...................................... 200 3. Selected applications ............................................................. 201 3.1 Sedimentation F F F (SdFFF) ....................................... 201 3.2 Thermal F F F (ThFFF) ................................................. 202 3.3 Electrical F F F (E1FFF) ................................................ 203 3.4 Flow F F F (FIFFF) ....................................................... 203 4. Comparison with SEC .......................................................... 204 5. Atomic spectrometry as element specific detection .............. 205 5.1 Literature ..................................................................... 205 5.2 F F F - I C P - M S for biological and environmental analysis ........................................................................ 210 5.2.1 Metal binding proteins ...................................... 210 5.2.2 Humic substances . ......... 211 5.2.3 Tissue and foodstuffs ........................................ 212 5.3 Quantitative analysis by F F F - I C P - M S ....................... 214 6. On-channel flow-fff preconcentration with atomic spectrometric detection ................................................................................ 215 6.1 Frit outlet ..................................................................... 216 6.2 Opposed-flow sample concentration ........................... 217 6.2.1 General overview and application .................... 217 6.2.2 On-channel matrix removal and pre-concentration ............................................................................ 221 7. Conclusion and future trends ................................................. 223
Acknowledgements ............................................................... 225 References ............................................................................. 226 C h a p t e r 5: Slurry sample introduction in atomic spectrometry : application in clinical and biological analysis ......................................... 237 1. Introduction ........................................................................... 237 2. Overview and nomenclature .................................................. 238 2.1 Slurry preparation ....................................................... 239 2.2 Particle size ................................................................. 240 2.3 Slurry concentration .................................................... 241 2.4 Chemical (matrix) modification .................................. 241 2.5 Calibration techniques ................................................. 241 2.6 Precision and accuracy ................................................ 242 2.7 Nomenclature .............................................................. 242 3. Slurry sample introduction .................................................... 242 3.1 Atomic absorption spectrometry (AAS) ..................... 243 3.1.1 Flame atomic absorption spectrometry(FAAS) .247 3.1.2 Electrothermal atomic absorption spectrometry (ET-AAS) .......................................................... 244 3.1.3 Flow injection techniques ................................ 246 3.2 Flame atomic emission spectrometry (FAES) ............ 247 3.3 Direct current plasma .................................................. 247 3.4 Inductively coupled plasma-atomic emission spectrometry (ICP-AES) ............................................. 247 3.5 Inductively coupled plasma-mass spectrometry (ICP-MS) ..................................................................... 248 3.6 Microwave-induced plasma-atomic emission spectrometry (MIP-AES) ............................................ 248 3.7 Atomic fluorescence spectrometry (AFS) .................... 249 3.8 Thermal vaporization (TV) techniques ........................ 249 4. Analytical figures of merit .................................................... 251 5. Practical applications of slurry sample introduction ............. 255 6. Conclusions ............................................ :.............................. 256 7. Suggestions for future studies ............................................... 257 Acknowledgements ............................................................... 258 References ............................................................................. 258 C h a p t e r 6: Application of laser-induced breakdown spectrometry in biological and clinical samples ............................................. 287 1. Introduction ........................................................................... 287 2. Fundamental studies .............................................................. 290 2.1 The interaction of a laser beam with target materials.. 290
2.2 2.3
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Laser-induced plasma production ............................... 293 Factors influencing plasma formation ........................ 295 2.3.1 Laser parameters ............................................... 295 2.3.1 A Influence of the irradiation wavelength.295 2.3.1 B Influence of irradiation energy ............. 297 2.3.2 Physical properties of the target material .......... 298 2.3.3 Ambient conditions ........................................... 300 2 . 3 . 4 Influence of electric and magnetic fields .......... 302 2.3.5 Plasma shielding ............................................... 303 2.3.6 Effect of sampling geometry ............................ 304 Excitation temperatures and electron densities ..................... 304 3.1 Excitation temperature calculations 304 3.2 Electron density calculation ......................................... 306 3.2.1 Electron number densities from stark broadening calculation .......................................................... 306 3.2.2 Electron number densities from Saha-Eggert ionization calculations ...................................... 307 3.3 Experimental results .................................................... 308 Spectral and analytical characteristics of LIBS .................... 310 4.1 Basic principles of LIBS ............................................. 310 4.2 Analytical characteristics ............................................ 311 Instrumentation ...................................................................... 314 5.1 Excimer laser and CO~ laser based LIBS .................... 316 5.2 Nd: YAG laser based LIBS instruments ..................... 316 5.3 Fiber-optic based LIBS instruments ........................... 316 5.4 Field instrumentation .................................................. 319 5.5 New approaches to LIBS ............................................ 322 5.6 Echelle spectrometer ................................................... 325 Applications ........................................................................... 326 6.1 Environmental applications ........................................ 327 6.2 Metallurgical samples ................................................. 333 6.3 Applications to liquids and solutions .......................... 339 6.4 Applications to aerosols and gases ............................. 342 6.5 Applications to non-metallic solids ............................ 343 6.6 Applications for advanced materials ............................. 345 6.7 Miscellaneous applications ......................................... 347 Conclusion ............................................................................. 348 References ............................................................................. 348 7: Application of graphite furnace atomic absorption spectrometry in biological and clinical samples ......................................... 361
~
Introduction ...................................................................................... 361 1.2 Spectroscopy ............................................................... 362 1.2.1. Introduction to atomic spectroscopy ................. 362 1.3 G F A A S analytical signal: absorbance ........................ 363 1.4 The nature of the transient G F A A S signal: m e c h a n i s m of atom formation in a graphite furnace ..................... 365 1.5 Instrumentation ........................................................... 366 1.5.1 Graphite furnace ............................................... 367 1.5.2 Graphite tube material and design .................... 368 1.5.3 Furnace heating cycle ....................................... 370 1.5.4 Methods of atomization .................................... 373 1.6 Sample preparation and sample introduction .............. 374 1.6.1 Liquids ........................................................... 375 1.6.2 Solids ................................................................ 376 1.6.3 Wet decomposition ........................................... 376 1.6.4 Combustion ....................................................... 378 1.6.5 Fusion ................................................................ 379 1.6.6 Solids analysis with slurry sampling (see Chapter 5 ) ................................................. 379 1.6.7 Direct solid sampling ....................................... 381 1.6.8 Laser ablation .................................................... 381 1.6.9 Preconcentration/separation methods ............... 381 1.6.9.1 Extraction ........................................ 382 1.6.9.2 C h r o m a t o g r a p h y ............................. 383 Flow injection analysis ................... 384 1.6.9.3 1.6.9.4 Other preconcentration/separation methods ........................................... 387 Metal speciation .............................. 387 1.6.9.5 1.7 Determination of elements by G F A A S ....................... 388 1.7.1 Applicability ..................................................... 388 1.7.2 Sampling, sample storage, and sample preparation ........................................................ 390 1.7.3 Quality control procedures ............................... 392 1.7.4 D e v e l o p m e n t of G F A A S methods .................... 393 1.8 Applications ................................................................. 396 1.8.1 Multielement continuum source G F A A S ......... 396 1.8.2 Determination of lead in blood by tungsten-coil AAS ................................................................... 397 1.8.3 Determination of arsenic and tin ...................... 398 1.8.4 Determination of c a d m i u m and zinc by double
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resonance laser-excited atomic fluorescence in an electrothermal atomizer ..................................... 400 1.8.5 Copper determination in biological materials by ETAAS using W-Rh permanent modifier .... 400 1.8.6 Determination of urinary lead, cadmium and nickel in steel production workers by GFAAS 401 1.8.7 Determination of platinum in clinical samples. 401 Conclusion .................................................................. 402 References ................................................................... 403 INDEX ....................................................................... 405
PREFACE
As of Volume 6, Elsevier Science has taken over the publication of this book series, previously published by JAI Press, Inc., CT, USA. Volume 6 was published as a special issue of Microchemical Journal, 2000, vol. 66, nos. 1-3, pages 1-172. The contents of the previous six volumes follow this Preface. This volume continues the tradition of the previous volumes with cutting-edge and current advances in atomic spectroscopy. A new development in the book series is that this volume and subsequent planned volumes have a focus in the area of atomic spectroscopy. This volume focuses on the application of atomic spectroscopy in biological and clinical samples. Where appropriate, the inclusion of other samples is provided to ensure complete coverage of a particular topic. Certain topics, e.g., LIBS in Chapter 6 are just beginning to find an application in this area and so its potential is discussed. Graphite furnace atomic absorption spectrometry (GFAAS) is well established and has a long use in this area. Chapter 7 discusses the technique and focuses on more recent applications A brief biography of all the contributors to this volume is given and a short abstract of each chapter of this volume is provided at the beginning of each contributed chapter. The editor of the book series (Joseph Sneddon) would like to thank the patience of all contributors and the reviewers for their excellent comments which have greatly enhanced this volume.
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Contents and Contributors to Volumes 1-6 in the Series
Volume 1 (1992) Chapter 1: Analyte Excitation Mechanisms in the Inductively Coupled Plasma Kuang-Pang Li, University of Massachusetts-Lowell, Lowell, Massachusetts, USA, and James D. Winefordner, University of Florida, Gainesville, Florida, USA. Chapter 2: Laser-Induced Ionization Spectrometry Robert B. Green and Michael D. Seltzer, Instrumental Chemical Analysis Branch, China Lake, California, USA Chapter 3: Sample Introduction in Atomic Spectroscopy Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA Chapter 4: Background Correction Techniques in Atomic Absorption Spectrometry Gerald R. Dulude, Thermo Jarrell Ash Corporation, Franklin, Massachusetts, USA Chapter 5: Flow-Injection Techniques for Atomic Spectrometry Julian F. Tyson, Department of Chemistry, University of Massachusetts, Amherst, USA Volume 2 (1995) Chapter 1: Laser-Excited Atomic and Molecular Fluorescence in a Graphite Furnace David J. Butcher, Western Carolina University, Cullowhee, North Carolina, USA Chapter 2: Electrothermal Vaporization Sample Introduction into Plasma Sources for Analytical Emission Spectrometry Henryk Matusiewicz, Politechnika Poznanska, Poznan, POLAND Chapter 3" Hydride Generation Techniques in Atomic Spectroscopy Takahara Nakahara, University of Osaka, Sakai, Osaka, JAPAN Chapter 4" The Excimer Laser in Atomic Spectrometry Terry L. Thiem, United States Air Force Academy, Colorado Springs, Colorado, USA, Yong-Ill Lee,
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Konyang University, Nonsan, Chungnam, KOREA, and Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA Chapter 5" Recent Developments in Analytical Microwave-Induced Plasma Robert C. Culp and Kin C. Ng, California State University, Fresno, California, USA Volume 3 (1997) Chapter 1: Plasma Source Mass Spectroscopy Andrew S. Fisher and Les C. Ebdon, University of Plymouth, Plymouth, Devon, England, UNITED KINGDOM Chapter2: Multielement Graphite Furnace and Flame Atomic Absorption Spectrometry Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA and Kimberly S. Farah, Lasell College, Newton, Massachusetts, USA Chapter 3: Direct Current Arcs and Plasma Jets Rudi Avni, Nuclear Research Center-Negev, Beer-Sheva, ISRAEL, and Isaac B. Brenner, Geological Survey of Israel, Jerusalem, ISRAEL Chapter 4: Direct and Near Real-Time Determination of Metals in Air by Impaction-Graphite Furnace Atomic Absorption Spectrometry Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA Volume 4 (1998) Chapter 1: Electrostatic Precipitation and Electrothermal Absorption Spectroscopy: A Perfect Combination for the Determination of Metals Associated with Particulate Spectroscopy Giancarlo Torsi, Clinio Locatelli, Pierluigi Reschiglian, Dora Melucci, and Felice N. Rossi, University of Bologna, Bologna, ITALY Chapter 2: Chemical Modification in Electrothermal Atomic Absorption Spectrometry Dimiter L. Tsalev and Vera I. Slavekova, University of Sofia, Sofia, BULGARIA
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Chapter 3" Recem Developments in Flow-Injection Atomic Spectroscopy Maria Delores Luque de Castro and L. Gameiz-Garcia, University of Cordoba, Cordoba, SPAIN Chapter 4: Determination of Mercury by Atomic Spectroscopy Joseph Sneddon and Mary Gay Heagler, McNeese State, Lake Charles, Louisiana, USA Volume 5 (1999) Chapter 1 9Speciation Studies by Atomic Spectroscopy Miguel de la Guardia, M.L. Cervera and A. MoralesRubio, University of Valencia, Valencia, SPAIN Chapter 2: New Types of Tunable Lasers Xiadeng Hou, Jack X. Zhou, Karl X. Yang, Peter Stchur, and Robert G. Michel, University of Connecticut, Storrs, Connecticut, USA Chapter 3: Developments in Detectors in Atomic Spectroscopy Frank M. Pennebaker, Robert H. Williams, John A. Norris and M. Bonner Denton, University of Arizona, Tucson, Arizona, USA Chapter 4: Glow Discharge Atomic Spectrometry Sergio Caroli, Oreste Senofonte and Gialuca Modesti, Instituto Superiore di Sanita, Rome, ITALY Chapter 5: Laser Induced Breakdown Spectrometry Yong-Ill L e e , Changwon National University, Changwon, KOREA, and Joseph Sneddon, McNeese State University, Lake Charles, USA Volume 6 (2000) Chapter 1: Capillary Electrophoresis Inductively Coupled Plasma Mass Spectrometry Vahid Majidi, Los Alamos National Laboratory, Los Alamos, New Mexico, USA Chapter 2: Thermospray S a m p l e Introduction to Atomic Spectrometry Xiaohua Zhang, Ding Chen, Rob Marquardt and John A. Korpchak, Southern Illinois University, Carbondale, Illinois, USA
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Chapter 3: The Real-Time Analysis of Gases by Direct SamplingMass Spectrometry: Elemental and Molecular Applications David J. Butcher, Western Carolina University, Cullowhee, North Carolina, USA Chapter4: Use of Atomic Absorption Spectrometry for the Determination of Metals in Sediments in South-West Louisiana James N. Beck, Nicholls State University, Thibodeaux, Louisiana, USA, and Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA Chapter 5" Field Instrumentation in Atomic Spectroscopy Xiandeng Hou and Bradley T. Jones, Wake Forest University, Winston-Salem, North Carolina, USA Chapter 6: Microwave Plasma Torch Analytical Atomic Spectrometry Wenjun Yang, Hanqui Zhang, Aimin Yu, and Qinhan Jin, Jilin University, Changchun, PR CHINA
Short Biography of Contributors to Volume 7
Joseph Sneddon is Professor in the Department of Chemistry at McNeese State University, Lake Charles, Louisiana. He attended the University of Strathclyde in Glasgow, Scotland obtaining a B.Sc. (honors) in Chemistry in 1976, M. Sc. in Instrumental Methods of Analysis in 1978 and Ph. D in Chemistry in 1981. He was a postdoctoral research fellow at the University of Strathclyde in 1980-81 and has served on the chemistry faculty at New Mexico State University, Las Cruces, New Mexico, California State University, Pomona, California, and University of Massachusetts, Lowell. He was Department Head at McNeese State University from 1992-1995. His research interests are in the general area of atomic spectroscopy, more recently in its application to environmental and biological samples. He has authored or co-authored over 140 papers and original articles in this area. He has edited several books, most recently Lasers in Atomic Spectroscopy (1997), Practical Guide to Graphite Furnace Atomic Absorption Spectrometry (1998), and Laser-Induced Breakdown Spectrometry (2000). He has been the editor of Microchemical Journal since 1990. Chapter 1 Marina Patriarca holds a Degree in Chemistry from the University of Rome, Italy and a M.Sc. in Medical Sciences from the University of Glasgow (UK). She currently holds the post of Senior Research Scientist at the Department of Clinical Biochemistry, Istituto Superiore di Sanit~ (ISS, Italian Institute of Health), in Rome (Italy), where she has been working as a Research Scientist since 1988. A large part of her research activity in the field of clinical biochemistry has been devoted to the development of methods for trace element analysis in human body fluids and tissues and investigations of their biological role in population studies. Further insight into human metabolism of trace elements has been obtained using stable isotopes as tracers in clinical studies, as part of the projects carried out during Dr. Patriarca's long-term collaboration with the Department of Pathological Biochemistry of the University of Glasgow (UK). Dr. Patriarca
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has authored more than 70 publications and has lectured in several international and national conferences and training courses on analytical and quality issues related to laboratory medicine. Antonio Menditto is a senior research scientist and Chief of the Section of Clinical Chemistry at the Department of Clinical Biochemistry of the ISS. Dr Menditto received his Degree in Medicine and Surgery from the University of Rome in 1984. He has presented over 80 lectures and seminars, and has published over 70 papers on various topics of biomedical research, human health, environmental toxicology, and laboratory medicine issues. He has served on the organizing and scientific committees of various national and international conferences. He has served on national and international environmental and human health committees including OECD and UNEP. Dr. Menditto and Dr. Patriarca have undertaken several activities for the promotion of metrology and quality assurance in the field of preventive, environmental and occupational laboratory medicine, among which the organization of the Italian national external quality assessment schemes (EQAS) for trace elements and metabolites of organic substances in body fluids, participation in the activities of the Thematic Network of European EQAS organizers in occupational and environmental laboratory medicine and collaboration to European Union projects for the certification of reference materials. Barbara Rossi received her Degree in Biology from the University of Rome I (Italy) in 1998. Since 1999 she collaborates with the Section of Clinical Chemistry, Department of Clinical Biochemistry at the ISS in the field of trace element analysis by atomic spectrometry and the promotion of quality assurance.
Chapter 2 Hiroaki Tao graduated from the Department of Chemistry of the University of Tokyo, Toyko, Japan in 1980. He received his Ph. D from the same university in 1986. He joined the National Institute of Advanced Industrial Science and Technology (AIST) in 1982. From 1993 to 1993 he was a visiting research fellow with the Institute for Environmental Chemistry, National research Council of Canada, where he worked with Dr. J.W. McLaren. H e has been the Group Leader of the Measurement Technology Group, Institute for Environmental Technology, AIST since 1999. His current research interests include elemental speciation using hyphenated methods such as gas chromatography-inductively coupled plasma-mass spectroscopy (GC-ICP-MS) and liquid chromatography-
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inductively coupled plasma-mass spectroscopy (LC-ICP-MS), sample preparation for atomic spectrochemical analysis, chemical sensors, and ETAAS. He is the author of about fifty publications including book chapters and research papers. He is a member of the editorial board of Bunseki Kagaku (Journal of the Japan Society for Analytical Chemistry) since 1998. Taketoshi Nakahara graduated from the Department of Applied Chemistry of Osaka Prefecture University, Osaka, Japan in 1965. He completed his Ph. D thesis at the same University in 1972. From 1976 to 1977 he was a visiting research fellow with the Department of Chemistry, Carlton University, Ottawa, Canada, where he worked with Professor of C.L. Chakrabarti. He was promoted to Associate Professor in 1985 and Professor in 1993 at the Osaka Prefecture University. His research interests include atomic absorption spectrometry, atomic fluorescence spectrometry with low temperature flames, atomic emission spectrometry with inductively coupled plasma and microwave induced plasma, inductively coupled plasma-mass spectrometry, and gas phase sample introduction techniques with vapor generation (e.g., hydride generation methods) for all kinds of analytical atomic spectrometry. He is the author of some one hundred and eighty publications including book chapters and research papers. Dr. Nakahara was the editor of Spectochimica Acta Reviews, Associate Editor of Applied Spectroscopy and a member of the editorial board of Spectrochimica Acta, Part B, and is currently a member of the editorial boards of Journal of Analytical Atomic Spectrometry, Atomic Spectrometry Updates, Canadian Journal of Analytical Sciences, Spectroscopy, and Microchemical Journal.
Chapter 3 Juan Manuel Marchante-Gay6n obtained a B.Sc. in Chemistry in 1990, and Ph. D in Analytical Chemistry in 1995 from University of Oviedo, Oviedo, Spain. He became an Assistant Professor at the University of Oviedo in 1995. His research interests and experience are centered mainly in the field of atomic spectrometry, with special emphasis in the areas of trace metal analysis and speciation in biological samples. He has published twenty papers. He is a member of the Spanish Society for Analytical Chemistry and Grupo Espectroquimica Espanol. Christina Sariego-Mufiiz obtained a B.Sc. in chemistry in 1995 from the University of Oviedo, Oviedo, Spain. She studied as an Eramus student at the University of Plymouth, Plymouth, United Kingdom in 1996. She started her Ph. D in 1997 at the University of Oviedo in the field of trace
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metal analysis and speciation in biological samples using inductively coupled plasma-mass spectrometry. Jose Ignacio-Alonso obtained a B.Sc. in Chemistry in 1980 and Ph. D in Analytical Chemistry in 1985 from the University of Oviedo, Oviedo, Spain. He was a postdoctoral research fellow at the University of Plymouth, Plymouth, United Kingdom between 1986 and 1987. He returned to University of Oviedo in 1987 as a postdoctoral fellow. In 1990 he became a scientific officer of the European Commission and was appointed to the Transuranium Elements, Joint Research Center, in Karlsruhe, Germany. After five years (in 1995) became a senior lecturer at the University of Oviedo, and in 1996 became head of Mass spectrometry Analytical Services at the University of Oviedo. His research interests and experience are centered mainly on the field of inductively coupled plasma-mass spectrometry, with special emphasis in the areas of trace metal speciation and Isotope Dilution Analysis, both for environmental and biological samples. In recent years, his research has concentrated on the development of semi-quantitative methods for trace metal analysis in environmental samples, the application of isotope dilution analysis for the analysis of biological materials and development and applications of interfaces for coupling gas chromatography to inductively coupled plasma-mass spectrometry for trace metal speciation. He has published around sixty papers. He is a member of the Spanish Society for Analytical Chemistry and fellow of the Royal Society of Chemistry and serves on the Editorial Board of Journal of Analytical Atomic Spectrometry Alfredo Sanz-Medel has been a Professor of Analytical Chemistry since 1982 at the University of Oviedo, Oviedo, Spain. After completing his Ph. D in 1973 at University of Zaragoza, Zaragoza, Spain in 1973, he was a postdoctoral research fellow in 1974 at Imperial College of Science and Technology, University of London, London, United Kingdom with Professor Tom S. West. He was an Assistant Professor of Analytical Chemistry at Complutense University, Madrid, Spain for four years and in 1978 joined the chemistry faculty at the University of Oviedo. He is the author of two hundred and forty publications, and several patents and books. He is a well-known speaker in his country and abroad about his group's research at the University of Oviedo. His research interests include three lines of analytical technologies; (a) new atomic detectors and methodologies for ultra-trace metals elemental analysis, particularly the use of plasmas (microwave induced plasma, glow discharge, inductively coupled plasmaatomic emission spectrometry and inductively coupled plasma mass spectrometry), (b) new molecular sensors, usually based on luminescence
xxiii
and fiber optic techniques for biological and medical applications, and (c) hybrid techniques for toxic metal analysis and speciation in biological and environmental samples, particularly the use of high performance liquid chromatography and capillary electrophoresis coupled with plasma detection. He was President from 1989 to 1999 of the Grupo Espectroquimico espanol and serves on the Editorial Board of Journal of Analytical Atomic Spectrometry, Microchimica Acta, and the Royal Society of Chemistry (United Kingdom) "Book Section" of monographs in Analytical Spectroscopy. Recently he has been appointed Associate Member of the Commission V of IUPAC. He also serves on the Editorial Board if ICP Information newsletter, Atomic Spectrometry Updates (RSC), Talanta, Anales de Quimica International and was a past member of the advisory board of Analytica Chimica Acta.
Chapter 4 Atitaya Siripinyanond is an analytical chemistry Ph. D student at the University of Massachusetts, Amherst since 1997. Her research (under the supervision of Professor Ramon M. Barnes) is focused on elemental speciation in biological and environmental samples using field-flow fractionation coupled to inductively coupled plasma mass spectrometry. She graduated with a B.S. in chemistry in 1994 and M.S in Chemistry in 1996 from Mahidol University, Bangkok, Thailand. She is supported by a fellowship from the Thai government funded through the Ministry of University Affairs. Ramon M. Barnes is Professor Emeritus of Chemistry at the University of Massachusetts, Amherst, where he served on the faculty since 1969. He has been conducting research on ICP and other discharges since 1968 when he spent one-year (1968-69) at Iowa State University, Ames, Iowa at a Postdoctoral Associate after receiving his Ph. D from University of Illinois, Urbana, Illinois in 1966. He serves as the chairman of the Winter Conference on Plasma Spectrochemistry, produced the monthly ICP Information Newsletter, and is director of the University Research Institute for Analytical Chemistry in Amherst, Massachusetts.
Chapter 5 Henryk Matusiewicz is Professor of Chemistry in the Department of Analytical Chemistry at Poznan University of Technology, Poznan, Poland. He received his Ph. D in 1973 and Dr. Sc (habilation) in 1987 in analytical chemistry from Poznan University of technology and University of Warsaw, Warsaw, Poland, respectively. In 1996 he was promoted to Professor of
xxiv
Chemistry. Since 1994 he has been the Head of the Analytical Chemistry Department at Poznan University of Technology. He was a Postdoctoral Research Associate at Colorado State University in 1975-1977 and the University of Massachusetts, Amherst in 1982-1984, visiting scientist at US Food and Drug Administration, Maryland, USA, Elemental Analysis Research Center in 1984-1985 and at NRCC, Institute for Environmental Chemistry, Canada in 1988-1996, and visiting professor at the University of Hanover, Germany, University of Dortmund, Germany, Max-Planck Institut fur Metallforschung, Germany, (1992), and University of Oviedo, Spain (1997).
Chapter 6 Yong-Ill Lee is an associate professor in the Department of Chemistry at Changwon National University, Changwon, Korea. He received a M.Sc. in Polymer Science in 1991 and Ph. D in Analytical Chemistry in 1992 from the University of Massachusetts, Lowell. He was a visiting research professor in the Department of Chemistry at Purdue University in West Lafayette, Indiana for 2000/2001. His main research interests are in analytical spectroscopy in general, laser and molecular spectroscopy and more specifically the development and application of new analytical techniques for atomic spectroscopy of advanced materials such as metals and ceramics. Recently he has started work on mass spectrometry in biological applications. He is a member of the editorial board of Microchemical Journal, Spectroscopy Letters and Applied Spectroscopy Reviews. Kyuseok Song is principal researcher of the Laboratory of Quantum Optics at Korea Atomic Energy research Institute (KAERI), Taejon, Korea. He received a M.Sc. in Physical Chemistry in 1982 from Korea University in Seoul, Korea and Ph. D in Physical Chemistry from Iowa State University in Ames, Iowa, USA. His major research interest has been in laser spectroscopy in general, analytical applications of atomic and molecular spectroscopy, and the development of new optical as well as mass spectroscopic techniques in the analysis of environmental samples. He has a strong interest in developing ultra-sensitive detection techniques for rare isotopes. He has authored over sixty scientific papers, six book chapters and a co-author of Laser-Induced Breakdown Spectrometry (2000). Joseph Sneddon see earlier
XXV
Chapter 7 David J. Butcher is professor in the Department of Chemistry and Physics at Western Carolina University, Cullowhee, North Carolina. He obtained a B.Sc. in Chemistry at University of Vermont at Burlington in 1984 and Ph. D in Analytical Chemistry from the University of Connecticut at Storrs in 1990 with Dr. Robert G. Michel. He is a member of the editorial board of Spectroscopy Letters, Microchemical Journal, and Applied Spectroscopy Reviews. He was the program chair of FACSS 2001. His research interests are in laser spectroscopy for chemical analysis, graphite furnace atomic absorption spectrometry, mass spectrometry, molecular spectroscopy and the application of these techniques to conifer forests and the environment in the western part North Carolina, Eastern part of Tennessee and southwestern part of Virginia. He is the co-author with Joseph Sneddon on the recent book, Practical Guide to Graphite Furnace Atomic Absorption Spectrometry (1998). Joseph Sneddon-see earlier
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Abstract of Chapters in Volume 7
CHAPTER 1 Use of Atomic Spectrometry (ICP-MS) in the Clinical Laboratory Since its introduction as an analytical technique, atomic spectrometry has found wide application in the clinical laboratory. More than 25 elements are important to human life, most of which present at trace or ultratrace levels. Several trace elements are routinely determined in body fluids and tissues for the diagnosis and monitoring of genetic diseases, nutritional deficiencies and occupational or environmental exposure. The choice of the method to apply for the determination of a specific trace element in a human sample requires a clear understanding of the clinical question and the relative performances and limitations of the available techniques. Inductively coupled plasma mass spectrometry (ICP-MS), the latest development of atomic spectrometry, has the capabilities for the fast and simultaneous determination of trace and ultratrace elements, with detection limits in most cases superior to graphite furnace atomic absorption spectrometry. High resolution ICP-MS can be used for the determination of most elements in body fluids and tissues, requiting only minimal sample pretreatment. Some of the interferences limiting the application of quadrupole ICP-MS to biologically important elements have been overcome using alternative methods of sample introduction, such as electrothermal vaporization and hydride generation, on-line chromatographic separation of interfering species and modified plasma conditions (cool plasma). New instrumental developments (collision/reaction cell technology) have been shown to reduce substantially the extent of major argide based interferences. Beside the determination of the total content of trace and ultratrace elements in clinical samples, the identification of their chemical species is necessary
xxvii
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in order to evaluate their bioavailability and relative toxicity. The on-line coupling of ICP-MS with separation techniques (HPLC, capillary electrophoresis) has been applied to the speciation of essential and toxic elements, such as As, Se and I, and to pharmacokinetic studies of metallodrugs. Stable isotopes are used as tracers in human studies to provide a direct assessment of the absorption, distribution and elimination of labelled compounds. In comparison with other techniques for the identification of isotope composition, ICP-MS allows faster sample throughput with. minimal sample preparation and it is therefore more suitable for studies of mineral metabolism. In addition, the development of ICP-MS reference methods based on isotope dilution can give an important contribution to the improvement of the quality and traceability of analytical data for trace elements in laboratory medicine.
CHAPTER 2 New Developments in Hydride Generation-Atomic Spectrometry Recent advances in hydride generation techniques in atomic spectrometry are overviewed. Fundamental research on novel hydride generation and chemical interferences and their elimination are described. Instrumental developments for speciation of the hydride forming elements, based on chromatographic or electrophoretic separation, post-column on-line sample pre-treatment and hydride generation followed by atomic absorption spectrometric or inductively coupled plasma-mass spectrometric detection are presented. Applications of these techniques in biological and clinical materials are also reviewed. Emphasis is placed on speciation.
CHAPTER 3 Analysis of Biological Materials by Double Focusing Inductively Coupled Plasma-Mass Spectrometry Inductively coupled plasma-mass spectrometry (ICP-MS) is (arguably) the most powerful detector in atomic spectrometry being the quadrupole mass filter the most popular analyzer in ICP-MS due to its relatively low cost and easy handling. However, the full potential of ICPMS cannot be exploited by conventional quadrupole-instrumentation because of spectral interferences. There are a variety of approaches by which such interferences may be compensated for in a practical analysis. However, the only general method to overcome limitations from spectral interferences is high mass resolution. Such high mass resolution can be obtained by Double Focusing-ICP-MS (DF-ICP-MS) instrumentation which combines a magnetic and an electric sector field analyzer. Although
xxix
available since 1988, DF-ICP-MS has not found widespread acceptance until recently, when the high cost of initial generation on instrumentation was considerably reduced with the introduction of a second generation DF-ICPMS instrumentation. This gave a strong impetus to the development of DFICP-MS applications in the analytical community. This is reflected in the increasing number of publications and an international conference devoted exclusively to high resolution sector field ICP-MS, and in general, a growing interest in the analytical performance of this technique. The aim of this chapter is to highlight the major areas of biological research where DF-ICPMS can provide an important contribution by reviewing both basic concepts of DF-ICP-MS and also recent developments in elemental analysis, isotope measurements and speciation of trace and ultratrace elements in biological and clinical samples. CHAPTER 4
Field-Flow Fractionation-Inductively Spectrometry
Coupled
Plasma-Mass
This chapter provides a current view of field-flow fractionationinductively coupled plasma-mass spectrometry (FFF-ICP-MS) applied to the biomedical, environmental, nutritional, and polymeric materials. Primarily the chapter is written to introduce practical information about FFF to the spectroanalytical chemist. The chapter begins with a section describing elemental speciation using chromatographic and non-chromatographic separations coupled with element specific detection techniques. A brief history of FFF and a general overview of different techniques in the FFF family follow. Four fields (i.e., sedimentation, thermal, electrical, and crossflow) that can be used for FFF are discussed. Selected applications of each FFF technique to biomedical and environmental samples are reviewed. After describing essential FFF features, (e.g., how it works, basic principles and physicochemical measurements, applications and application ranges, instrumentation and optimization), FFF is briefly compared with sizeexclusion chromatography especially for macromolecular characterization. The application of atomic/mass spectrometry as elemental detection for FFF is treated next with an emphasis on speciation in ICP-MS. A novel feature of flow-FFF (flFFF) for on-channel pre-concentration with either a frit outlet or opposed-flow sample concentration also is described. In the final section, FFF-ICP-MS is identified as an important growth area both for practical applications and research. Selected presentations made at the international conferences are presented.
XXX
CHAPTER 5 Slurry Sample Introduction in Atomic Spectrometry: Clinical and Biological Analysis
Application in
A short overview of slurry sample introduction in atomic spectrometry is presented, including both fundamental and physical considerations of slurry sample introduction. Methods for slurry sample introduction into atomic absorption spectrometry (AAS), inductively coupled plasmas (for atomic emission and mass spectrometry-AES, and MS, respectively), microwave induced plasmas (MIP-AES), direct current plasma (DCP), atomic fluorescence spectrometry (AFS) are reviewed and critically evaluated and the performance of these atomic sources for real sample determination is evaluated. Brief comparisons of detection limits for analytical atomic spectrometric methods that utilize slurry sampling as presented in most published reports are discussed. Finally, the literature on the application of the selected results from an updated application of slurry sampling techniques to clinical and biological materials are discussed and presented. CHAPTER 6
Laser-Induced Breakdown Spectrometry : Potential in Biological and Clinical Samples When the output from a pulsed laser is focused on to a small spot of a sample, an optically induced plasma, called a laser induced or laser ablated plasma is formed at this sample surface. This will occur when the laser power density exceeds the breakdown threshold value of the surface. When the laser created plasma is used as a source for atomic emission spectrometry, it is frequently called laser induced breakdown spectrometry (LIBS). In recent years this technique has attracted a great deal of interest from the analytical community, particularly in its application to situations where it clearly has advantages over conventional analytical atomic spectroscopic techniques. This chapter will give a brief overview of the basic principles and instrumentation for LIBS and will focus on the application to clinical and biological samples.
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CHAPTER 7
Application of Graphite Furnace Atomic Absorption Spectrometry in Biological and Clinical Samples Graphite furnace atomic absorption spectrometry (GFAAS) is an established, and reliable analytical technique for trace and ultra-trace metal determination in may samples. Despite its wide acceptance and maturity, it continues to find new applications. This chapter will primarily focus on these new applications as it applies to clinical and biological samples. Following a brief overview of the technique and instrumentation, the results and recent applications will be presented.
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Chapter 1
Use of atomic spectrometry (ICP-MS) in the clinical laboratory Marina Patriarca, Barbara Rossi, and Antonio Menditto Laboratorio di Biochimica Clinica, Istituto Superiore di Sanit/l, viale Regina Elena 299, 00161 Rome, Italy I. INTRODUCTION Of the elements in the periodic table more than 25 are important to human life [1-2]. Along with the constituents of organic matter, electrolytes (Na +, K +, Ca 2+, Mg 2+ and Cl) and trace elements (Co, Cr, Cu, Fe, I, Mn, Mo, Se and Zn) participate in biochemical processes necessary to maintain life and perform essential functions. Some of the non-essential elements pose threats to human health, when exposure occurs at the workplace or from the general environment. Other potentially toxic elements are deliberately administered as a therapy in severe illnesses (e.g., Li+ in manic depression, Pt complexes in cancer, Au in rheumatoid arthritis, Bi in gastric ulcer) and other metallodrugs are under development [3]. Alterations of the concentrations of trace elements in body fluids and tissues occur in pathological conditions, nutritional deficiency, following drug administration and as a result of occupational or environmental exposure. Measurements provide essential information for the prevention, diagnosis and monitoring of diseases and therapy [4-6]. Besides the determination of the total content of trace and ultratrace elements, increasing interest is paid to speciation, as the occurrence of an element in separate identifiable forms affects its bioavailability, metabolism and/or toxicity [7]. Atomic spectrometry has been applied in laboratory medicine since its introduction as an analytical technique. Electrolytes and some trace elements can be detected in biological fluids and tissues by simple and rapid flame atomic spectrometry methods. The development of electrothermal atomic absorption spectrometry (ETAAS) in the late '70s improved the detection limits by 10 to 100-fold and allowed the investigation of the biological role of trace and ultratrace elements [8]. The limitations of ETAAS" single-element analysis, time consuming
ADVANCES IN ATOMIC SPECTROSCOPY Volume 7, ISSN 1068-5561
1
Copyright 9 2002 Elsevier Science B.V. All rights reserved
M. PATRIARCA, B. ROSSI, and A. MENDITTO
procedures, prone to severe matrix interferences, stimulated research for alternative sources of sample atomization, which led to the development of plasma source atomic spectrometry [9]. Most elements are efficiently ionised in an argon plasma and may be detected on the basis of their optical emission or mass spectra. Both inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS) can perform fast, multielement determinations and can be coupled on-line with separation techniques [9-10]. However, the concentrations of most trace elements (or their species) of interest in laboratory medicine are beyond the detection limits of ICPOES. In addition to its superior detection power, ICP-MS also has the ability to determine the isotopic composition of a sample, requiring much less sample pretreatment than other mass spectrometric techniques. Over the last 15 years, several innovative developments have occurred in ICP-MS and led to improved performances, especially for elements below mass 80, determination of which is affected by severe interferences. Although the cost of purchasing and rtmning an ICP-MS, including special laboratory requirements and trained personnel, is still high, the number of applications of ICP-MS in laboratory medicine is continually and rapidly increasing. According to manufacturers [11], the number of ICP-MS insmnnents sold each year is increasing, owing to the improvement in instrument performance, the development of simplified operating procedures and userfriendly software and the ever-increasing demand for the measurement of a greater number of elements and their chemical species at lower concentration in several fields of human activities. These considerations suggest that the use of ICP-MS in the clinical laboratory will continue to grow. Several papers and books have reviewed technical aspects of atomic spectrometry [9-10, 12] and its biomedical applications [8, 13-16]. Updates of new applications to clinical and biological samples are published regularly[ 17-20]. This contribution aims to give an overview of the use of atomic spectrometry in the clinical laboratory, with special reference to applications of ICP-MS. The suitability of different analytical atomic spectrometric techniques for their application to specific problems in the clinical laboratory is discussed. The technical features of ICP-MS and its latest development are reported in more detail. Particular attention is paid to the application of atomic mass spectrometry in laboratory medicine for the determination of trace element species for clinical and biological monitoring purposes and for the study of the metabolism of elements in humans using stable isotope tracers. Other important developing areas are elemental speciation and the use of isotope dilution ICP-MS (ID-ICP-MS) for the establishment of the traceability chain for the results of measurements of elements in biological materials.
Atomic Spectrometry in the Clinical Laboratory
2. ATOMIC SPECTROMETRY LABORATORY
TECHNIQUES
IN THE
CLINICAL
2.1 Requirements for trace element analysis in the clinical laboratory The specimens to be analysed are typically blood, serum or plasma and urine. The analysis of hair, nails and tissues (generally liver and bone biopsies) may also be required. Other less common specimens include cerebrospinal fluid, saliva and seminal fluid. Typical reference ranges for the electrolytes and trace elements most commonly measured in the clinical laboratory are reported in Table 1 [6, 8, 14-15, 21-26]. The determination of one or more elements may be necessary in the same sample and the level of accuracy required may vary from simple screening procedures to confirmatory tests. The choice of the method to apply to the determination of a specific trace element in a human sample requires a clear understanding of the clinical problem and the performances and limitations of the available techniques. Beside appropriate detection limits and reliable analytical performances, the size of sample needed, the throughput time, the ability for multielemental analysis, the level of operator skill needed and capital and running costs are all important variables to be taken into account. The fimess for purpose of analytical methods applied in laboratory medicine should be evaluated according to clinical needs [27-29]. It is generally agreed that criteria based on clinical efficacy or biological variation should be used whenever possible to set standards of analytical performance [30-31]. Recommendations of experts (individuals or groups), standards set by laws, regulatory bodies or organisers of external quality assessment schemes (EQAS) and the state of the art, judged from the results of interlaboratory comparisons or the literature, can be used when other data are not available [30-31]. In Table 2, standards of desirable performance for imprecision, bias and total allowable error, estimated from available data on intra- and interindividual biological variation, are reported for some electrolytes and essential trace elements [32-33]. Table 3 shows the maximum allowed errors, in terms of deviation from the target values, set by the organisers of the Italian EQAS for trace elements in biological fluids [34].
M. PATRIARCA, B ROSSI, and A. MENDITTO
Table 1 Reference ranges for electrolytes and trace elements in biological fluids [6,8,14-15]
Element
Specimen, unit
i?
General population
A1
Sa'",
As
U b, ~tg L l U, lag L1
1-30 1-60
Au
S/P, lag L "l
3000. This may be a problem, if the size of the sample available is small or the element concentration low [ 16, 76]. In Q-ICP-MS, collision or dynamic reaction cells provide a new means to overcome polyatomic interferences and are expected to find wide application in the future. With current Q-ICP-MS insmmaents, some interferences can be eliminated by choosing methods of sample introduction, which allow the chemical or physical separation of the analyte from the matrix. Interferences from ArC1+ on the determination of As and Se in biological matrices is generally avoided by generating their volatile hydrides, which can be transported directly into the plasma. Sample introduction via ETV is effective in reducing most oxide-based interferences, such as those affecting clinically important elements (e.g. Fe, Se). The choice of the fimaace temperature programmes must be tailored for the specific analyte/matrix at hand, thus posing limitations to multielemental analysis in biological matrices and to internal standardisation other than by isotope dilution. Alternatively, the analyte can be separated from interfering species prior to analysis by off- and on-line chromatographic methods, provided that the dilution factor does not prove prohibitive, or by simple chemical procedures. Most transition elements form stable neutral chelates, which can be extracted in organic solvents. This approach removes interferences from major components of biological matrices, such as Na, K and C1, and increases sensitivity by concentrating the analyte, but it is rather time-consuming and prone to contamination. As most transition elements are likely to be co-extracted, isobaric interferences with other overlapping isotopes are not excluded.
AtomicSpectrometryin the ClinicalLaboratory
17
Table 7 Typical isobaric and polyatomic interferences affecting the determination of elements of clinical or toxicological importance in biological matrices
Element
24Mg 27A1
Interferences
12C2+
285i
14N2+spread 14N2+, 12C160+
31p 32S 39K 4~ 51V 52Cr 55Mn 56Fe 58Ni 58Ni 59C0 63Cu 64Zn 67Zn 7~ 75As 77Se 79Br 8~ 95Mo
14N16OH+ 1602+ 38ArH+ 4~ + 35Cl160+ 4~ 36Ar160+, 36S160+, 35Cl16OH+ 4~ + 4~ 58Fe+ 4~ 42Ca160+, 41Cal6OH+, 4~ 43Ca160+ 4~ +, 31p1602+ 32S1602+ 35Cl1602 + 35C12+ 4~ +, 38Ar37C1+ 4~ + 4~ 40AF2+ 79Br160+
97Mo
81Brl60+
Required resolution ~,1600 ~ 1600 ~1600 ~970 ~ 1800 ~5700 ~193000 ~2600 ~3000 ~3000 ~2500 ~28000 ~3000 ~3000 ~3000 ~--~000 ~,~4000 ~6000 ~7800 ~9000 ~ 10000 ,~10000 ~12000 ~18000
Operating the plasma under 'cold' conditions, i.e. at low power with a high nebuliser flow rate, reduces the occurrence of Ar-based interferences and elements with low ionization energy, such as K, Ca and Fe can be determined with lower detection limits [123]. However, there is little effect on non Ar-based interferences and additional polyatomic species may be generated from complex matrices when operating under these conditions [ 124].
18
M. PATRIARCA, B. ROSSI, and A. MENDITTO
The addition of other gasses (e.g. N2, He or CH4) to the carder gas [ 125-126] or organic solvents, such as methanol or butanol, to the diluent [127-128] have also been shown to reduce some of the Ar-based interferences and enhance the response for elements with ionisation energy in the 9-11 eV range, such as As, Se and Hg.
4. 3.2 Non spectral interferences Other, non spectral, interferences occur in ICP-MS which are due to physical influences on the sample introduction system, fluctuation of the plasma stability affecting the intensity of the signal, differences between samples and standards affecting sample introduction, sample transport and the properties of the plasma. These can be generally controlled using an internal standard. Ideally the element chosen as intemal standard should be monoisotopic, unlikely to be found at concentrations above the limit of detection in the sample itself, unaffected by spectral interferences and of mass and ionisation energy close to those of the elements of interest. In practice, compromises are inevitable. In multielemental analysis more than one internal standard is often necessary, spread across the mass range scanned: Sc, for masses up to 80 amu, Rh or In, for the range between 80 and 150 amu and Ir, for masses above 150 amu are frequently used.
4.4 Applications For several elements, ICP-MS provides lower detection limits than other techniques and for some, it is the only technique capable of achieving reliable results at the concentrations found in biological fluids and tissues. Even when these concentrations are much lower than those associated with known clinical effects, establishing their ranges for unexposed subjects is essential to serve as references in the assessment of exposure to environmental pollutants. It should be borne in mind that became of this higher power of detection, control of contamination is of the utmost importance, requiring in some cases, specialised handling facilities, such as Class 100 laboratories. Reagents should be of the highest purity available, as the actual limits of detection depend on the blank values. Calibration is generally performed using aqueous standards, however, matrix matched standards should be used if matrix effects are not negligible. All analytical procedures should be thoroughly validated, whenever possible by analysing matrix-matched certified reference materials. Quadrupole ICP-MS instnunents are by far the most popular, due to their lower cost and easier handling. Several elements of interest in environmental and occupational medicine can be measured by this technique in blood (Be, Cd, Hg, Pb, Sb) sertun (Ba, Bi, Cd, Cs, Hg, Pb, Mo, Sb, Sn) or urine (Ba, Be, Bi, Cd, Hg, Pb, Sb, Sn, Te, Th, T1, U, W) with limits of detection between 1 and 500 ng L "~ [14, 113-115]. At least fourteen elements of clinical interest (B, Br, Ca, Cu, 54Fe, I, K,
Atomic Spectrometry in the Clinical Laboratory
19
Li, Mg, 98Mo, Pt, Rb, Sr, Zn) can be reliably determined in serum samples, in some cases with an appropriate choice of isotopes and mathematical corrections of interferences [113-115, 129]. Generally, procedures for the simultaneous determination of elements in blood, serum or urine have been implemented, which significantly increase the efficiency of routine laboratories and exploit the amount of information obtained from a single sample. The interferences affecting the determination of As in urine and Se in serum and urine can be overcome using HG-Q-ICP-MS, after complete digestion of the sample, with limits of detection of about 0.1 lag L 1 for As and between 0.6 and 1.8 ~tg L "1 for Se in serum. Alternatively, accurate determinations of As in urine can be performed with mixed plasmas (N2) or diluting the samples with 1% ethanol [ 130]. A similar direct procedure for the measurement of Se in serum, whole blood and erythrocytes by ICP-MS aiier dilution with a solution containing 1.0% v/v butan- 1-ol has been described [ 128, 131 ]. With careful selection of analytical masses, chemical modifiers and temperature programmes, elements overlapped by polyatomic interferences can be measured reliably by ETV-Q-ICP-MS and improved absolute limits of detection can be obtained for other elements. S e l e n i u m (77Se and 82Se) was determined in 10 ~tl of 20-fold diluted blood serum, with a limit of detection of approximately 0.1 lag L 1 and a long-term precision of 3.8% [132]. Aluminium, Ti and V were measured in serum with limits of detection of 0.7, 0.4 and 0.1 ~tg L "1 [133]. Improved limits of detection have been reported for the determination of Mo in serum (10 ng L "l) [134], Pt in urine (1 ng L "l) [135] and rare earth elements in urine (from 1 to 10 ng L -l) [136]. Became of the variability of transient signals, the best results are achieved using isotope dilution for standardisation. The determination of Cd and Pb in twine by ETV-ID-ICP-MS yielded limits of detection of 20 and 5 ng L l, respectively, and precision < 11% [ 137]. Several papers have reported the simultaneous determination of trace elements in autopsy tissues by Q-ICP-MS. However, the level of accuracy varies from element to element, depending on concentrations and the analytical conditions adopted. According to an investigation carrried out on certified reference materials (CRMs), including Bovine Liver NIST SRM 1577b and Human Hair NCS DC 73347, Ba, Cd, Cr, Cu, Li, Mn, Ni, Na, Pb, Sr, and Zn can be reliably determined in sample aliquots ranging from 1 to 50 mg, with limits of detection ranging from 0.02 to 0.38 ~tg g-i [ 138]. The analysis of foetal and paediatric tissues is one of the more demanding as the concentrations of most elements are low. In a study of 157 paediatric livers, the concentrations of Ag, Cd, Co, Pb and Sb were measured by QICP-MS aiter pressurised digestion with HNO3, with limits of detection ranging from 0.14 to 3.8 ng g-1 wet mass [ 139].
20
M. PATRIARCA, B. ROSSI, and A. MENDITTO
Since several elemems of clinical and toxicological interest suffer from more or less severe interferences in Q-ICP-MS, the applications of SF-ICP-MS to biological and clinical samples are growing, especially when lower limits of detection are required [15, 140]. A recent report described the simultaneous determination of 57 elements in 10-fold diluted digested whole blood. The limits of detection were less than 1 ng L "1 for Cs, Dy, Er, Eu, Gd, Ho, Ir, Lu, Pr, Pt, Re, Sm, Ta, Tb, Th, Tm, U and Yb; between 1 and 10 ng Ll for Ag, Au, Ce, Cd, Ga, Hf, La, Nb, Nd, Rb, Sc, Sb, T1, Y and W; and between 10 and 100 ng L1 for Be, Ba, Bi, Co, Ge, Hg, Li, Mn, Mo, Pb, Sn, Sr, Te, V and Zr. Higher limits of detection were observed for As, Cu, Cr, Ni and Ti (between 0.1 and 0.5 lag L l) and A1, B, and Se (between 1 and 2 ~tg L "l) [141]. Similar results were obtained for Co, Cr, Mo and Ni in whole blood simply diluted with (1+9) with a solution containing Triton X- 100, EDTA and NH3 [ 142]. Other papers [ 143-145] have reported the determination of several elements of clinical interest in diluted (1+4, 1+7) serum, including Ag, A1, Ca, Cd, Co, Cr, Cu, Fe, Mn, Mo, P, Pb, Rb, S, Si, Sn, Sr, Ti, U and Zn. Paediatric reference ranges (~tg g~ creatinine) for the concentrations of Cr (0.07-0.76), Ni (0.20-1.23) and V (0.02-0.22) in urine were estimated from 131 subjects by means of SF-ICP-MS. Urine was diluted 1+19 and spectral interferences were resolved using a resolution factor of 3000 [ 146]. Cadmimn, Cu, Pb, and Zn were measured simultaneously in 10-fold diluted urine, using a higher resolution factor (3000) for the determination of Cu and Zn, whereas Cd and Pb were measured at lower resolution (300) for better sensitivity. The trace elements most frequently determined for clinical and toxicological purposes (AI, As, Cd, Cr, Co, Cu, Hg, Mn, Ni, Pb, Sb, Se, T1 and Zn) could be measured simultaneously in 10-fold diluted urine, as well as in digested blood and 100-fold diluted serum [147]. In addition, the determination of several essential (Co, Cr, Cu, Fe, Mn, Ni, Se, and V) and non essential (Ag, AI, As, Au, Pt, Sc and Ti) elements in human milk by SF-ICP-MS has been reported [148]. 5. STABLE ISOTOPE DIAGNOSIS
TRACERS:
A TOOL FOR RESEARCH AND
5.1 Background Both radioactive and stable isotopes are used in biochemistry and medicine as tracers of mineral metabolism in animal models and humans. Tracer studies provide a direct assessment of the absorption, distribution and elimination of electrolytes or trace elements, which is necessary in several branches of medicine, such as nutrition, toxicology, pharmacology and clinical biochemistry. Stable isotopes are a safer alternative to radioactive isotopes as tracers for studies in humans. The safety of their use allows the extension of investigations to all
Atomic Spectrometry in the Clinical Laboratory
21
population groups, including children and pregnant women, for whom critical information on mineral and trace element metabolism is often lacking. The availability and sensitivity of analytical techniques for their determination is however a key issue for the exploitation of the potential of stable tracers for human studies. Inductively coupled plasma mass spectrometry has several advantages for such studies because of the minimal sample preparation required and the high sample throughput. Precision of the measured isotope ratios is acceptable for most biological tracer studies (typically , 9 54 -.] 2~ >, 55
ZZ >.
New Developments in Hydride Generation - Atomic Spectrometry
67
reactions (interferences) observed in the bulk aqueous phase. This vesicle-assisted Cd hydride generation has been utilized to flaker increase the AAS sensitivity for Cd by in situ trapping in a graphite fumace [44, 45, 47]. This highly positive effect of vesicles for HG could be also combined with their potential in vesicular HPLC separations for CA speciation in human urine and fish cytosols [49]. Sanz-Medel et al. also reported on the cold vapor generation ofmonoatomic Cd~by reduction of Cd2+solution with NaBH4 using vesicles [43]. They proposed the mechanism shown below for the cold vapor generation of Cd: BH4"+
3H20 + H + --+ H3BO3 + 8H"
(1)
8H- + Cd 2+ ~ CdH2 + 2H2 +2H +
(2)
CdH2
(3)
-->
Cd 0 + H 2
where H. is "nascent" hydrogen. Since CAH2 is volatile, this should be the transportation mechanism for cadmium. However, at room tempeman~, during transport, the volatile hydride which is formed would decompose according to reaction 3, and the resulting Cd ~ which is not volatile, would detx~it all along the connecting tubing. 1his decomposition is more efficient at higher temperatures. In any case, the great excess of H2 formed by reaction 2 would prevent the complete decomposition of CdH2. Thus, at least a ceaain amount of CdH2 would ultimately reach the absorption cell. Once there, according to reaction 3, a substantial proportion of CdH2 seems to be able to form monoatomic Cd~ Although this cold valor would not be thermodynamically stable, the lifetime of monoatomic Cd~ appears to be sufficiently long to be measured. 1his provides the basis for the AAS measurement of Cd at room tempemau~. Guo and Guo also developed HG (cold vapor generation) using thiourea 'and Co as calalysts for the reaction, resulting in an increase in sensitivity [50, 51]. The detection limits obtained with this method were 20 pg m1-1 by AAS and 8 pg m1-1 by AFS. Hwang and Jiang used the same media for HG and determined CA in urine by
68
H. TAO and T. NAKAHARA
ICP-MS [52]. An isotope dilution (ID) technique was used to alleviate the depressing interference from the concomitant elements, qhe detection limit was 26 pg m1-1.Bennejo-Barrem et al. generated the hydride in the absence of organic w.zction media by adding Ga as a catalyst, resulting in a detection limit of 4 pg ml~ with the in situ trapping / ETAAS [53]. Cmnara and cxr-workers showed that it was not ner.essary to add surfactants and metallic species as catalysts in order to generate the volatile Cd species [54, 55]. They achieved a detection limit of 50 pg ml~ with cold vapor AAS and applied the p ~ u r e to file analysis of a number of standard reference water samples; but, the method of standard additions was needed to alleviate interference from the concomitant elements, qhey later extended the applications to the analysis of waste water and sewage sludge, and interference from co-existing metals such as Cu, Pb, Ni ar~ Zn was overcome via the addition of potassium cyanide. (Caution: This must have produced considerable amounts of hazardous HCN on merging with the acid carrier stream.) 3. ADVANCES OF METHODS OF ATOMIZATION 3.1. Atomization interferences in the gas phase In addition to graphite fumaees, diffusion flames and quartz tubes are also employed to atomize hydrides for AAS. In quartz tubes, there are two types: 'flameless' extemally heated quartz tubes and flame-in-tube a t o ~ . Atomization interferences are intimately coupled to the mechanism of hydride atomization and to the fate of free atoms in the observation volume of the atomizer. Two types of atomization interferences are currently proposed: 1) A radical population interference due to the depletion of hydrogen radical population in the atomizer. 2) An analyte decay interference due to acceleration of the decay of free analyte atoms in the atomizer. Welz and Stauss investigated the mechanism of atomization interferences in the 'flameless' externally heated quarlz tube atomizer [56]. They reported that in the batch HG system, the radical deficiency constittaed the main reason for the low tolerance of other hydride-forming elements and that in the flow injection HG system, the prevailing mechanism was analyte
New Developments in Hydride Generation - Atomic Spectrometry
69
decay interference due to sm'hc~ alterations catts~ by the deposition of the interferent in the quartz tube atomizer. D'Ulivo and ~ dina investigated the mechanism of atomization interefere~ in flame-in-mt~ and in diffusion flame atomizers [57]. For this purpose, they developed a hydride atomizza" which was able to operate in lhese two modes. Liquid phase interferences were eliminated by using a twin-channel continuous-flow hydride generator. They repotted that only the analyte decay interference was significant in unheated flame-in-tube atomizers and ditSasion flames. 1hese atomizem, which were characterized by a relatively large supply of oxygen, were inherently resistant to the radic~ population interference since hydrogen radic~ production was proportional to the oxygen supply. They also repotted that the removal of free analyte atoms at high analyte concentrations or in the presence of the interfetent was dominated by the reaction with polyatomic species and/or particles formed at high c o ~ t m t i o n s of analyte and interferent atoms. 13~dina and D'Ulivo developed an argon-shielded highly fuel-rich hydrogen-oxygen diffusion microflame, refened to as a flame-in-gas-shield atomizer, for AFS [58]. The sensitivity was at least 2 times higher and the noise was lower lhan the miniature diffusion flame. Sensitivity .was controlled by interaction of the analyte (Se) with atmospheric gases. Analyte free atoms were removed from the observation volume by chemical re,actions with oxygen penetrating fi'om the ambient atmosphere. No significant quenching effect due to the intem~on of excited Se atomic levels with nitrogen or hydrogen was found. I~ dina et al. investigated the mechanism of hydride atomi~tion and the fate of free atoms in the miniattae diffusion flame [59]. Selenium hydride was used as a model for other hydrides. The spatial temtmature distribution was highly inhomogeneous ranging from 150~ to 1300~ The entire flame volume was a c a ~ y a cloud of hydrogen radicals, which maintained the analyte in the flee atom slate, sirm~ hydrogen radicals which were formed in outer zone of the flame ditfused to its cooler inner regions. Tesfalidet et al. determined hydrogen radicals in a miniaturized oxygen/hydrogen flame, which is similar to a flame-in-tube atomizer, by means of electron spin resonance (ESR) spectt~copy to investigate the
70
H TAO and T. NAKAHARA
atomization mechanism [60]. By using this technique, they were able to monitor the production of hydrogen radicals in the hydride atomization step and the decline and consumption of these radicals when AsH3 was intaxtuced into the flame as a model compound. These results provided the direct experimental evidence needed to support the proposed mechanism for the atomization of hydrides, i.e., that atomization is brought alx~ by hydrogen radicals. Matott~ek et al. measured the cross-sectional distribution of free Sb atoms in quartz tube atomizers by AAS using a CCD camera [61]. They confirmed that the highest fi~ atom concenWations were found near the tube axis, decreasing towards the walls, in the ~ e a t e d flame-in-tube atomizer. In the extemally h~aed atomizer, the most widely used in routine analysis, the free atom d'~tribution was much more homogeneous compaw.d to the unheated atomizer under analytical conditions, although pronounced inhomogeneity was obtained at high Sb concentrations in a roll-over part of the calibration curve, qhis was explained on the basis of free atom decay on the surface ofpolyatomic particles formed at high analyte concentrations. Grinberg et al. developed an externally heated quartz tube atomizer which diffened from the usual T-tube design only in having 5 holes drilled along the length of the optical tube, which was heated by the acetylene-ak flame [62]. q'hey investigated mtmml hydride-forming element interference and found that the holed quartz tube a t o ~ was able to induce larger tolerance limits for the interference in 9 of the 16 mtmml interference possibilities studied. D&lina and Matou~ek developed a multiple microflame quartz tube atomizer which consisted of two concentric tubes [63]. The inner tube had multiple tiny orifices over its length in the wall and the otaer tube, which was devoid of orifices, was externally h~/ed as the conventional quartz tube atomizer. They demonstrated that lifts set-up reduced the poor resistance to atomization interferences and the unsatisfactory linearity of calibration graphs. Numemm fundamental studies have been ~orted on atomization mechanism and many improvements ofqtmrtz tube atomizers and diffusion flames have been made in the past few years. By using newly developed techniques such as ESR spectroscopy, fia~er progress on the atomization
New Developments in Hydride Generation - Atomic Spectrometry
71
mechanism and elimination of atomization interferences is expected.
3.2. In situ Trapping HG / electrothermal atomic absorption spectrometry The most atWactivc advantages of the in situ trapping HG / ETAAS technique are the very low detection limits and the decrease of the kinetics interferences in HG and interferences in the atomizer. Matusiewicz and Sturgeon presented a review of this methodology [6]. Palladium is both a well-recognized chemical modifier in ETAAS and an efficient collector for the trapping of volatile hydrides. One of the main problems of a Pd-treated atomizer is that a Pd modifier solution must be applied before every hydride trapping nm because of its thermal instability during the atomization step. Ni et al. used a graphite atomizer coated with Ag to trap the hydrides of Se and Te [64]. The advantage in this approach is that atomization occurs at 1800~ lower than that for a Pd-coated surface (2000~ thereby increasing the lifetime of the tube. Shuttler et al. proposed the in situ trapping of hydrides of As, Se and Bi in a graphite tube coated with a Pd-Ir modifier as a "permanent modifier", which allowed up to 300 complete trapping and atomization cycles [65]. Other "permanent modifiers" tested included Ir sptater-c~ated tubes [66], carbide coatings onto which Ir had been deposited [67-69] and other carbide-forming elements [70, 71]. Haug and Yiping investigated two groups of trapping reagents, i.e., carbide-forming elements (Zr, Nb, Ta or W) and noble metals Or, Pd-Ir) [70]. The effective trapping of germane was possible on Zr-coated tubes and more than 400 complete trapping and atomiz~ion cycles were possible, k-coated graphite tubes allowed trapping at lower temt~'ature but the signals were small and the stability was low, compared with those for the Zr coating. C r a t ~ et al. also investigated a graphite tube crated with Zr [72]. The Zr coating employed was relatively stable; and, once formed it withstood about 80 firings without any significant change in the efficiency of hydride collection. Tsalev et al. evaluate~ an Ir-Zr-treated and k-W-treated platform as a permanent modifier and found that Ir was much more promising than Pd, being an efficient thermal stabilizer for numerous volatile elements
72
H. TAO and T. NAKAHARA
during more than 800 firings [67]. Although Ir was better stabilized on a W-treated platform than on a Zr-treated one, the vaporization and atomization temperature for volatile analytes were also higher and double peaks were observed for B i and Te, when an k-W-treated platform was ttsed, and therefore, an Ir-Zr-treated platform was the most promising. Tsalev et al. also performed an optimization study for HG and collection and investigated the behavior of some organometallic species of As, Sn and Se [68, 69]. Optimum HG conditions differed substantially for As(m), As(V), MMAA and DMAA, unless L-cysteine was added. Organoelement species of As, Sn and Se were thennMly stabilized in a similar manner on both Ir-Zr- and k-W-treated platforms, the least stable species being selenomelhionine and trimethylselenonium. Do~ekal et al. presented a table with available publications conceming the p r e ~ e n t of the graphite surface (Pd and/or Ir modifier) and the resulting efficiencies of riG and trapping till 1995 [73]. Efficiencies of riG and in situ trapping in the transversely heated graphite fumace with a L'vov platform by using Pd modifier were studied for selenium hydride, arsine and stibine by means of the radioWac~rs of 75Se, 76Asand lz2Sb. This ra~otmcer study proved that optimum conditions for H2Se were achieved with a broad range of Pd modifier mass, trapping t e x t u r e , carder gas flow rote and capillary distance. This is very important for routine applications of in situ trapping of hydrides to the determination of hyddde-fomaing elements by AAS, as variations in these parameters cannot negatively influence accuracy. Murphy et al. reported a comparison of the applicability oflr- and Zr-coated graphite tubes to simultaneous multi-element (As, Sb, B i, Se and Te) hydride trapping and found that Ir was well suited to this application [74]. They also developed a purification method for the NaBH4 solution using an on-line and off-line active alumina column. The purification is very important because the ~ detection limit is not limited by the instrumental sensitivity but by impurities in the NaBH4 reagent. HG and in situ trapping on the inner surface of the graphite tube, treated with Pd or Ir, was coupled with the determination by furnace atomic nonthennal excitation spectrometry (FANES) [75] or ICP-MS [76, 77], as
New Developments in Hydride Generation - Atomic Spectrometry
73
well as by ETAAS. 1hese methods were validated using standard reference materials of biological tissues and seawaters. Marawi et al. attempted to me strips of W, Ta, Mo and Re coated (electroplated/sputtered) with Pd as platforms inside the graphite ftmaace to trap the hydrides [78]. Scanning electron microscopy (SEM) showed a smoother Pd layer coveting the metallic substances when compared with the graphite. The Pd-sputteted W platform showed a superior performance over tt~ other platforms. In situ trapping with an untreated graphite furnace was coupled wilia the determination by Ar and He MIP-AES [79, 80]. A number ofpal~rs on flow injection (FI) / HG coupled to graphite fumace AAS with the in situ trapping of hydrides has been reported [72, 81-86]. Burguera and Burguera [81] and Burguera et al. [87] ttsed the Fleitmann reaction, in which arsine was generated by the reduction of arsenic with metallic aluminum in a basic medimn, instead of using NaBH4 in HC1. Walcerz et al., using a Pd-coated graphite furnace, reported that TritonX-100, added together with the Pd solution, improved sensitivity and reproducibility, likely by promoting a more even distribution of Pd over the s~ of the graphite tube [85]. Willie developed FI / on-line photo-oxidation / HG / in situ trapping / ETAAS system for the "fl~ order" speciation of As in biological tissue and urine samples [86]. The in situ trapping method was also applied to the detenuimtion of Ge in garlic [82], Sn in hair and serum [83], Pb in biological samples and ionic organolead compounds [84, 88], and Se and As in highly minemliz~ water [89]. Liao and Li trapped indium hydride into a pre-heated graphite ~ a c e coated with Pd and obtained greatly improved sensitivity for the determination of In by HG/AAS [90]. Tyson et al. investigated the effect of smile volume on the limit of detection in FI / HG / in situ trapping / ETAAS to evaluate the claim that a decrease in the limit of detection might be achieved by increasing the sample volume [91]. They observed that as the sample volume was ino'eased, the detection limit improved significantly up to a volume of 500 pl, but for volumes larger than 1000 lal no ~ r significant improvement was obtained.
74
H. TAO and T. NAKAHARA
4. CHEMICAL INTERFERENCES IN LIQUID PHASE AND PRE-REDUCTION Interference effects, especially by wansition metals, are widely realized to be very severe. A number of papers have attributed interferences to the capture and decomposition of hydride by finely dispersed concomitant metal precipitates [92-94] or metal borides [95-97]. Measures taken to minimize such interferences include use of masking agents, matrix isolation, and the introduction of flow-injection and membrane-based gas liquid separator systems. Wickstrom et al. reported on an approach to minimization of transition metal interferences for H2Se generation [98]. They ttsed complexing agents, such as EDTA and diethylenetriamine~taac~ic acid (DTAP), and reduced selenite to selenide with NaBH4 in an alkaline medium, followed by acidification of the solution to produce H2Se. The reduction of transition metal ions could be avoided before the hydride was released from the liquid phase, and high concentrations of interfering Wansition metal ions which were present in the solution were acceptable without causing interferences, 8000 mg 1-1for Ni2§ and Co2+, 5000 mg 1-1for Cr3§ 500 mg 1-1for Fe3+, and 20 mg 1-1 for Cu2§ Uggerud and Ltmd [99] and Bowman et al. [100] investigated the use of thiourea as an agent for the pre-reduction and masking of interferences in the multi-element detemfination of As, Sb, Bi, Se and Te by HG/ICP-AES and of As, Sb and Se by HG/ICP-MS, respectively. Martinez~ria et al. made a comparative study of the magnitude of the interference in different inorganic acid media during the determination of Sb by HG/AAS [101]. They proposed a scheme, which accounts for all the different interference mechanisms. In particular, the me of conditional reduction potentials, and of their variation with pH, provided a basis for deducing, in a general way, whether or not a particular chemical species was capable of producing interference. L~ysteine has proved to be very effective reagent in both reduction of interferences and enhancement of signals. In addition, L-cysteine is fairly
New Developments in Hydride Generation - Atomic Spectrometry
75
soluble in water, has low toxicity and has little smell compared with most other thiol compounds. The optimum concentration of acid required for the procedure can be reduced, resulting in a reduction in the acid-derived blank contribt~ons to the analyte response. Brindle and coworkers previously reported that Lcysteine s ~ s f u l l y reduces matrix interferences in the determination of As, Sb, Ge and Sn when HG techniques are used [102-105]. The pentavalent forms of Sb and As are ttst~y ~duced to their trivalent oxidation state before their determination by HG, and KI alone or mixed with ascorbic acid is most frequently ttsed for that purpose. However, KI can only be ttsed in strong acid media, typically 5-6 M HC1, and KI solutions are unstable and must be prepared fresh daily. For these reasons it would be desirable to find an alternative reagent for the pre-reduction of As(V) and Sb(V) to their lrivalent states and as an interference releasing agent. Chen et al. demonstrated the utility of L~ysteine over KI as an efficient pteteducing agent for As(V) and Sb(V) [106, 107] and Brindle et al. showed that L-cysteine could be ttsed for on-line reduction in a continuous-flow system [108]. Welz and Sucmanov~i optimized file analytical parameters for using L~ysteine as a preteducing and releasing agent in FI / HG / AAS [109, 110]. They showed that L-cysteine provided greater freedom from interference and much better stability of solution with low concentrations ofanalyte and the reagent consumption and particularly the acid concentration were significantly lower than KI, which pemaitted a change in the me of highly corrosive solutions to solutions of low acidity and low toxicity. Tolerance limits (less than 10% interference) of at least 250 and 500 mg 1-~ were found for Ni and Cu, respectively, in the presence of 1% L-cysteine, in the determination of Sb. For the determination of As, the corresponding tolerance limits were 200 mg 1-1 for Ni and more than 1000 mg 1-1 for Cu. Only 100 mg 1~ of Cu could be tolerated when KI was used. Lugowska and Brindle investigated the nature of precipitates formed by the reaction ofFe01), Ni(/I) and Co01) with NaBH4, which interfere with the determination of Se by HG, by potentiomelric titration, direct current plasma atomic emission spectrometry and positive ion FAB mass spectrometry [111]. The precipitates obtained by the reaction of acidic
76
H. TAO and T. NAKAHARA
solutions of Fe0I) with NaBH4 did not contain boron, whereas precipitates obtained fi'om Ni0I) and Co01) contained metal (55-68%) and boron (2.6-4.3%) in a ratio of 3-5 91.1he two hypotheses presented in the litemane [97, 112] can both be supported by the results of this work. (i) The hypothesis that the hydride is not formed explains the mechanism of the interferences in terms of the prevalerr.e of the reaction of NaBH4 with M(I1) (the higher rote and NaBH4 consumption, where MOO is the transition.metal ion) over the reaction of NaBH4 with IT and in terms of changing the mechanism of the HG under these conditions. (ii) The hypothesis conceming the decomposition of the previously formed hydride by the precipitates is based on the thermodynamic instability of H2Se (the potential of Se(0)/H2Se couple for 10-6 M H2Seis alx)ut -220 mV) in the presence of the boride-like species (steady redox potentials between 0 and -250 mV). The oxidation of HzSe during the formation of the boride-like species is exemplified by equation (4) for the reaction of the precipitate of the boride: 4Ni2++ 2BH4 + 3H2Se+ IT --~ 2Ni2B+ 3Se + 7.5H2
(4)
Interference in HG have mostly been studied with regard to total metal determination, but some have been investigated for speciation using HG / cryogenic trapping / C~ / AAS. Sulfur compounds and pigments have been sttspected of having depressive effects on HG [ll3]. Martin et al. investigated the effect of different organic comlxxmds (organic pollutants, organic solvents, complexing agent and humics) and a mixture of 14 inorganic compounds on organotin speciation (MMT, MBT, DBT and TBT) by HG / cryogenic trapping / C~ / AAS [114, 115]. Organic compounds had little effect on signal suppression for any of the organotin species. Interference effects by inorganic elements were found to be significant, but they could be successfully controlled by the use of masking agents such as EDTA and L-cysteine. Le et al. [116] and Guo et al. [ 117] reported that pre-reduction of the As species by marion with Imysteine equalized the sensitivities of the technique to As which was present as A s ( ~ , As(V), MMAA and DMAA.
New Developments in Hydride Generation - Atomic Spectrometry
77
Brindle and Le proposed that enhanced performance in the presence of Imysteine results from the formation of an H3B-SR intermediate from the reaction between Lmysteine and NaBH4 which is a more efficient reductant than NaBH4 alone [104]. ~B NMR spectra showed evidence of formation of an intermediate slxxfies, BH3SR-. On the other hand, Howard proposed that the enhancement has more to do with file formation of the reduced arsenic-cysteine complexes than to the formation of a super-reductant such as H3B-SR- [ 118]. In the conventional NaBH4 trMuction of As species those arsenic oxy-aniom which contain arsenic in the pentavalent state must fLrst be reduced to the tfivalent state. R,As(O)(OH)3~ + I-I+ +BH4" --~ Rd~OH)3-n + H20 + BH3
(5)
(in most cases, R is a methyl group and n ranges li'om 0 to 3.) A subsequent reaction with the NaBH4 converts the As compot~ to the corresponding arsine: Rdks(OH)3.. + (3-n)BH4 +(3-n)H + ~ ~ 3 - .
+ (3-n)BH3 + (3-n)H20
(6)
The pH characteristics of the NaBI-h reaction lead to the conclusion that for the re~.cfion to p ~ rapidly, the target species must not be present in solution in the form of a negatively charged species. This indicates that the arsenic oxy-anions must be fully protonated if they are to be conveaeA to arsine. Acid dissociation constants of several As and Se species are given in Table 3 and the pH dependency of arsine generation yield from various arsenic oxy-anions is shown in Figure 4. Since the pK1 for arsenic acid is 2.3 the reaction must therefore be carried out at very low pH and 1-2 M HC1 is typically used. However, in the case ofthiol compounds such as L-cysteine and thioglycollic acid (denoted as T-SH), it is hypothesized that strong acid conditions are tmnecessary, as the As species are pre-teduced from the pentavalent to the trivalent state prior to the NaBH4 reduction step. The standard oxidation potential of the cysfine-cysteine system is 0.074 V [69].
78
H. TAO and T. NAKAHARA
Thus, it is possible to reduce As(V) and Sb(V) to their trivalent states, and MMAA and DMAA to their trivalent thiolates" 2T-SH + Rd~O)(OH)3-n~ T-S-S-T+ Rnms(OI--I)3.n+ H20
(7)
The trivalent species can then react to give As0ID-thiol complexes: RnAs(OH)3.n + (3-n)T-SH~ Rdks(-ST)3-n+ (3-n)H20
(8)
The three co-ordinate As(m)-thiol complexes are both uncharged and less sterically hindered to attack by NaBH4. 1his would lead to the potentially faster and more uniform production of mines. On the other hand, L~ysteine also reduces Sn(IV) to Sn(ff), Se(IV) to Se(0) and Te(IV) to Te(0), and therefore, Se and Te cannot be detemfined. Moreover, interference by Se on Ge and Sb may ~ m e stronger presumably due to coprecipitation of these analytes with the elemental form of the Se. Howard and Salou investigated the pre-reduction of As(V), MMAA and DMAA with a range of sulfur compounds and found that pre-reduction with thioglycollic acid was more rapid than with L-cysteine and was better suited to continuous flow pre-reduction procedures [ 119]. The pre-reduction of Se(VD to Se(IV) is generally accomplished by heating the analyte in the presence of HC1, usually at an acid strength of 6 M. Hill et al. investigated the kinetics of Se(VI) reduction and reported that the activation energy for the reduction of Se(VI) to Se(IV) was calculated to be 90.4 kJ moll using 6 M HC1 [120]. This result indicates that the full reduction of Se(Vi) to Se(IV) may be achieved in as short a time as 6 min at 70~ Brindle and Lugowska explored mild conditions for reduction of Se(VI) to Se0V) and repotted that the most effective pre-reduc~t for Se(VD was bromide ion in acidic solutions [121]. They concluded that bromide ion was about 19 times more effective than chloride ion under the same conditions and that the reduction of Se(VI) was complete in 2 M HBr solution atter 2 min of boiling.
New Developments in Hydride Generation - Atomic Spectrometry
79
A~ENATE + ARSENIT-~ @.SEN,T 1.2-
amenite[ 0.8
-
.,
',,,
(D
:=0.4 m
\,
,....,.
MeAsO(OH ":, ", - .
arsenal 0.0
-I
-2.0
_.,
0.0
\ \
..p=Kp. / " .pK ~ ._- "IPK' "~_K~_ . ,~-.--~ ~--~- , 2 4 6 8
pH Figure 4 pH dependency of the amine yield obtained from the reaction of a number of arsenic oxy-anions wilh NaBH4. Small vertical arrows indicate the pH values at which pH=pK, for each acid. Reproduced by permission of the Royal ~ of Chemisa3' with acknowledgement to
Table 3 Acid dissociation constants of some acids ,,
,,|
_
,
,
,,,
,
Abbreviation
Acid ,|,
,
pK
,,
As(O)(O~
.......
AsiI As(V)
(CHs)ns(OXOH~
MMA
(CH3)As(O)OH Se(O)(OH)2
DMAA
Se(O)~(OH~
Se(VI)
Se(W)
....
P K 1 = 9.2" pK1 =2.3 pK 2=6.8 pK3 = 11.6 pK 1 = 4.0 pK 2 = 8.6 pK = 6.3 pK 1 =2.5 pK2 = 7.3 pKl=-3(estimate) pK2 = 2.0
80
H. TAO and T. N A K A H A R A
5. HYPHENATED TECHNIQUES
The biological and toxicological effects of an element often d ~ d on its chemical form or oxidation state. The determination of total element concentration does not provide this vital information. Coupling chromatography to an element-specific detection method such as AAS, ICP-AES and ICP-MS can pen~t the discrimination of various species. HPLC is usually the method of choice as a separation technique rather than GC because the latter requires a previous derivatization step to produce volatile substances, which is not always feasible. Recently, capillary electrophoresis (CE) has attracted extensive attention, ~ e of its high separation capability. Unfortunately, the detection limits obtained by simply coupling HPI~ or CE to AAS and ICP-AES have been less than adequate for speciation at trace level in te~ samples. The hydride generation technique has been used as the most successful melhod for improving the sensitivity of these hyphenated methods. Since the oxidation state and chemical forms which can form volatile hydrides are limited, it is also necessary to incorporate the on-line conversion system from non-hydride forming ~ i e s [e.g., AB and Se(VI)] to hydride forming species [e.g., As(ill) and Se(1V)] into the HPI~ / HG / AAS or CE / HG / ICP-MS systems in order to expand the applicability of these mefl~ods to a variety of species. 5.1. HPLC / on-line treatment / HG / atomic spectrometry A number of investigations c o n ~ g on-line oxidation systems, assisted by thermal [122, 123] or microwave (MW) heating [86, 124-129] or by UV in~ation [130-138], have been carried out for the determination of non-hydride forming organoarsenie species. Rieei et al. had previously reported on the on-line K2S2Os-HC1 oxidation step of p-aminophenyl arsonate incorporated in an ion chromatography / HG / AAS system [139]. L6pez et al. employed thermooxidation to b~lk down or~'x)arsenie eompotmds [122]. The t h e r m o - ~ o r consisted of a loop of PTFE tubing dipped in a
New Developments in Hydride Generation - Atomic Spectrometry
81
powdered-graphite oven heated to 140~ The thenno-conversion etticiencies were above 96%. On-line photooxidation combined with HG/AAS for the determination of organoarsenic eon~unds originated from the work by Atallah and Kalman [140], although on-line photoo~'dation itself had been used previously in the field of voltammetry for heavy metals [141] and in colofimetry for total organic carbon (TOC) and total nitrogen (TN) [142]. This photooxidation was also applied to organotins, since they give rise to hydrides of restricted volatility. These organotins could be converted to inorganic Sn(IV) by UV inadiation, which then gave stannane when reacted with NaBH4 [143]. The UV degradation of the As species in the presence of persulphate pemaits the tmasformation of all As sixties into As(V) to be cznied out in a short time. Rubio et al. reported on the detennimtion of the six arsenic species [As0ID, As(V), MMAA, DMAA, AB and AC] by HG/ICP-AES after HPLC separation and on-line photooxidation and reported that the detection limits (2~) were 7.9 ng As ml~ for AB and 6.1 ng As mll for AC [130]. The same technique has been ~klgorted for As speciation in marine biological materials [132, 135, 144]. Zhang et al. developed the technique of argon segmented flow in the post-column eluent, and obtained a substantial improvement in chromatographic resolution [135]. Guo and Baasner employed 'knotted' VIFE tubing reactor to reduce peak broadening [145]. Slejkovec et al. reported a dtml As speciation system, which combined HPLC and purge and trap (PT) / C~ separation with AFS detection [134]. Using HPI~ / UV photooxidation / HG / AFS, it was possible to sepm~ up to six As species with detection limits of ca. 0.5 ng ml-~As (100 lal injected). Using selective HG / PT / C~ / AFS, up to four As species with detection limits of ca. 2.5 pg ml-l (for 100 ml-sample size) could be determined. A limitation in the UV inadiation method would be the use of a mobile phase of organic character in HPLC separation, which could be decomposed during the photooxidation procx~ and, thus, the yield of analytes oxidation would be decreased significantly. Rubio et al. also reported on a HPLC / photo~duction / HG system using ICP-AES or QCAAS (Quartz Cuvette Atomic Absorption v
82
H TAO and T NAKAHARA
Spectrometry) as detection methods for Se(IV) and Se(VO determination [146]. The detection limits (2o) were 6.8 ng ml ~ for Se(IV) and 16 ng ml~ for Se(V1), when using ICP-AES, and 15 ng mll for Se(IV) and 33 ng ml"l for Se(VI) when using QCAAS. Separation was performed using an anion-exchange column and phosphate buffer as the mobile phase. The eluate from the column entered the photore,actor, where it underwent UV inadiation for 120 s by 15 W low-pressure Hg lamp. Effects ofthe pH ofthe medium and the presence of various reducing agents on the teAuction efficiency fi'om Se(VI) to Se(IV) were investigated. Reduction efficiency was highest in water and in 1% NaOH without the addition of any reductant, showing that neutral or alkaline media are the most suitable. A reduction efficiency in excess of 50% was obtained. Vilan6 and Rubio extended this approach to the determination of Se(IV), Se(VI), selenocystine (SeCys) and selenomethionine (SeMet) [147]. 1he detection limits (3o) for Se(1V), Se(VI), SeCys, and SeMet were 0.6, 14.5, 0.9, and 5.9 ng ml1, respectively. The method was validated by analyzing two water certified reference materials, in which only Se(1V) was detected. Although they concluded that, after photoreaction, all the Se species were converted to Se(IV), the conversion efficiencies from Se(VI), SeCys and SeMet to Se(IV) were not given in this paper. On-line MW oxidative digestion instead of photooxidation has been used for the conversion of organoarsenic compounds to As(V) by the reaction with KES2Os-NaOH. Compared with photooxidation, MW digestion appears to be subject to less interference from organic substances, which penrfits the use of a wider variety of mobile phase in HPLC. However, bubble formation sometimes leads to irregular flow rates and increased baseline noise with the MW digester. The cooling loop is n ~ a r y to condense gas bubbles and to prevent water vapor from entering the AAS detection unit [137]. On-line microwave aeamaent has also been used for pre-reduction fi'om Se(VI) to Se0V) by the reaction with HCI. Pitts et al. reported that the detection limits (3o) were 0.2 ng ml1 for Se(1V) and 0.3 ng mll for Se(VI), respectively, by using HPI~ / MW reduction / HG / AFS system [148].
New Developments in Hydride Generation - Atomic Spectrometry
83
Cobo-Femfi.ndez et al. reported on an HPLC / MW oxidation / MW reduction / HG / AAS system for the determination of Se(1V), Se(VI) and tfimethylselenonium ion (TMSe+) [149]. In this case, TMSe+ was first oxidized to Se(V1) with KzS2Os-NaOH and then reduced to Se(IV) by HC1 in a microwave oven. G6mez et al. extended this technique to the detem~ation of Se(IV), Se(VI), TMSe +, SeMet and SeCys [150]. M a r c h a n ~ y 6 n et al. ttsed a reverse-phase column modified with a vesicular mobile phase ofdodecylammonium bromide (DDAB) to separate Se(IV), Se(V1), SeMet, SeCys and selenoethionine (SeEt) [151]. Since only Se(IV) forms H2Se, the prior transformation of other Se compounds into Se(IV) is required. For this purpose, they and Gon~ez-laFuente et al. [152] used a MW-assisted one-step p r e ~ e n t by KBrO3-HBr, and Ellend et al. [153] used a MW-assisted one-step pretreatment by KBr-HC1. Since the use of acetonitrile, methanol or a vesicular fonning reagent, which are typically used in reverse-phase HPI~, sometimes presents problems owing to their organic nature, the mobile phase must be an appropriate medium for MW-assisted pretrealment and HG. Gon~ez-laFuente et al. repoaexl on the detemalna"tion ofSe(IV), Se(VI), SeMet, SeEt and SeCys in urine by HPI~ / MW prear.aanent (KBrO3-HBr) / HG / ICP-MS (double-focusing) [154]. A double focusing ICP-MS was operated in the 'low resolution' mode, to increase the sensitivity. They obtained a sensitivity 23-59 times higher than that obtained with a quadmlx~le ICP-MS. However, the limits of detection were only 1-8.7 times ~ than those found with the quadmt~le ICP-MS due to a serious increase in background noise from polyatomic ions of Ar and from Se contamination of the reagents. Tsalev attempted to evaluate three techniques, i.e., a thermostated bath (TB), a microwave-assisted digestion (MWD) and UV irradiation (UV) [3]. The efficiency in digesting organoelement compounds was ranked as follows: MWD .~UV + heating .~UV >> TB. For routine analysis HPI~ / on-line pretreatrnent / HG / AAS appears to be the more affordable instrumental approach over HPLC coupled with more expensive detectors such as ICP-MS or electrospray ionization MS.
84
H. TAO and T. NAKAHARA
5.2. CE / HG / ICP-AES (or ICP-MS) The key point to s u ~ f u l on-line coupling of CE with ICP-AES or ICP-MS is the design of the interface. The i n t e ~ should be able to provide the minimum band broadening of CE peaks. Since CE may only provide O ,..,.
NIST SRM2670,
Urine: digested with HNO 3 + H202; reduced with IICI in closed Teflon vessel 184 by MW digester. Urea was useful to eliminate NOx fumes, which was
urine
absorbed in the digest and interfere with IIG/AAS. Recovery ofTMSc § SeMet and SeEt added to urine was 96.5-105%. ScMet and SeEt were unstable during MW heating used to reduce Se(VI) to Se(IV). Such a MW reduction procedure should be cautiously used to distinguish Sc(VI) from Se(IV) in the matrices which might contain organosclenium conq~ounds.
t~ ,'=t O
,.7
total Se
isotope dilution /I IG/N 2 MIP-MS
10 pg lnl l
NIES SRM
Serum: added with Se spike; digested with IINO 3 and 11202. Interferences of
No.4 freezedried human blood serum
Ar-associated polyalomic ions on 78Se and 8~ MS.
191
Se(IV), total Se llG/in still Irapping 18 pg lnl 1 for 2 ml /ETAAS sample volume
orgauoselenium For total Se determination, selenosugar was completely decomposed to (selenosugar) inorganic Se by treatment with K2S2Os in a water bath at 85~ for 15 min oral nutrition without UV irradiation.
Se(IV), Se(VI), HG/in situ trapping 60 pg ml 1 for 1 ml Salllllh' Vllilllile TMSc +, ScMcl, /I~TAAS SeCys, ScPur, (corrcspoding to 3 ug total Se ml l for urine)
NIST SRM2670, urine
Urine (1 ml): digested with 10 ml of HBr + 0.5 ml of 0.35 M KBrO3 at 150 ~ C filr 2 h; destroy excess bromine by adding hydroxylamine hydrochloridc; diluted to 50 ml with 10% HCI.
189
total Se
blood serum, On-line ultrasonic-assisted wet digestion with H2SO4-HCIO4-|INO 3. The SRM Seronorm oxidation state of Se from the digested SeMet was +4. A cross flow nebulizer 116 worked as gas/liquid separator and allowed the simultaneous determination of other elements of clinical interest.
183
on-line ultrasonicassisted digestion /HG/ICP-AES
Se(IV), Se(VI) MW pre-reduction /HG/AAS
So(IV), Se(Vl), I I(;/AI:S TMSe +, SeMet, SeCys, SePur, total Se
5 ng mi 1 tor 300 ~tl sample volume
1.0 ng ml ~ for Se(IV) citric fruit an,l 1.5 ng ml 1 for Sc(VI) lor 300 lal sample volu me
{I.5 and l.{I ng ml l lor blood serum, 100 lal original urine urine, NIST and serum sample SRM 2670 wfl u me, respect ivcl y
Se(IV), SeMet HPLC(ion-exchane) 0.73 ng ml "l for /IlG/N2 MIi'-MS
juices, geothermal waters
Se(IV) and 8.7 ng ml "1 tor SeMet for 100 ~tl sample volume
urine
o~
were eliminated in N2 MIP-
195
MW-assisted thermoreduction of Se(VI) to Se(IV) with 12 M HCI. A knotted 196 reaction coil was used.
Urine (1 ml): digested with 10 mi of llBr + 0.5 ml of 0.35 M KBrO 3 ;it 122 ~ C for 90 min; destroy excess bromine by adding hydroxylamine hydrochloride; diluted to.50 ml with 10% HCI. For the analysis of samples like serum with a protein and fatty material content higher than urine, preliminary dissolution with HNO3 was necessary lbr quantitative recoveries.
185
192 Column, Hamilton PRP-X100 anion-exchange column (250 x 4.1 m m x 10 ~tm); Mobile Phase, 15 mM phosphate buffer (pll 7.0). With N2 MIP-MS, the polyatomic interference related to Ar was eliminated, and the major isotopes of 7SSe and S~ were used for the analysis. SeMet directly generated wdalile DMSe and DMDSe when reacted with NaBII4 and I ICI. No pretreaiment of SeMet prior to HG was necessary.
,.q 9 ,..q Z >
New Developments in Hydride Generation - Atomic Spectrometry
97
Se(IV) and Se(VI) in less than 90 min using 0.01 M Br2 in 48% HBr at a 122~ solution tempema~ [185]. The paxedure was s u ~ f u l l y adapted to the digestion of urine and blood serum samples, although a preliminary dissolution step with HNO3 was necessary to obtain quantitative recoveries for serum. The redox-buffer properties of the HBr-Br2 solution were originally employed to convert all the inorganic Se species into Se(1V) with the aim of determining the total inorganic Se in environmental samples [186, 187]. D'Ulivo et al. also investigated the mechanism of breakdown of organoselenium compounds in a HBr-Br2 digestion system by using HG and C~, both coupled with atomic f l u o ~ c e detection, polarography, and 1H and 77SeNMR spectrometry [188]. The roles played by Br2 and HBr in the conversion of organoselenium into inorganic Se(IV) were identified as (i) the oxidative addition of Br2 to divalent selenium to form bromoselenonium intermediates and (ii) the dealkylation of selenonium compounds by the bromide ion. The fact that, at the end of the HBr-Br2 digestion, the selenium is converted into Se(1V), and the abserr.e of potentially interfering acids such as HNO3, HC104 or H2SO4, make this digestion method attractive for the determination of total Se not only by HG/AFS but also by other methods such as C~ and polarography. Tyson et al. retorted on an off-line sample preparation method using HBr-KBrO3 for total Se determination in urine and found that for TMSe§ it was necessary to increase the digestion temperature to 150~ for 2 h to increase its percentage recovery to above 90% [189]. Marchante-Gay6n et al. [151] and Gonz~ez-laFuente et al. [152] detem~ed Se(IV), Se(VI), SeMet, SeCys and SeEt in urine by HPLC / MW prelw..alment (KBrO3-HBr) / HG / AAS (or ICP-AES, ICP-MS). Gon~ez-laFuente et al. reported on the determination of Se0V), Se(V1), SeMet, SeEt and SeCys in urine by the same HPLC / MW pretreatment / HG system using double-focusing ICP-MS [154]. G6mez et al. detemfined Se(IV), Se(V1), TMSe +, SeMet and SeCys in urine by HPLC / MW oxidation (K2S2Os-NaOH) / MW reduction (HC1) / HG / AAS [190]. They reported that Se(VI), SeMet and TMSe + were stable in the cleaned-up urine for at least two days. However, significant losses of Se(IV) were observed,
98
H. TAO and T NAKAHARA
probably due to its co-adsorption into the colloids that were formed with time and 60% of SeCys was transformed in 5 h to an unknown Se organic compound. Therefore, to avoid losses of the Se species studied, the fresh urine had to be cleaned-up and analyzed on the day of collection, especially if the s m i l e potentially contained Se0V) or SeCys. They concluded that most Se excretion occtmvd in the first 12 h alter ingestion of the sample. Ingested Se(VI) was partially excreted and partially transformed and excreted as a species that behave as SeCys. SeMet was completely transformed and excreted to species that behaved as Se(VI) and SeCys. Ohata et al. developed a method for the accurate detefinination of total Se in blood sennn by HG coupled with isotope dilution analysis using high-power nitrogen microwave-induc~ plasma mass spectrometry (N2 MIP-MS) [191]. Interferences of Ar-associated polyatomic ions on 78Se and 8~ were eliminated in N2 MIP-MS. Chatterjee et al. detennined Se(IV) and SeMet in urine by HPLC / HG / N2 MIP-MS [ 192]. Flow injection (FI) employing on-line microwave (MW) reduction followed by HG / AAS [193] or AFS [194] has been developed for the differential deten'nination of Se(IV)and Se(VI). "I'he Se(V1)concentration was given as the difference between total Se concentration and Se(IV). A mini-column was used in FI / MW reduction / HG / AFS for retention and specific elution of Se(IV) and Se(V1) with fomaic and hydrochloric acids, respectively [194]. A detection limit (30) of 0.04 ng mll and reproducibility of 1%). This means that urine and serum should be diluted 1:5 or even better 1:10 to prevent this effect. Higher salt concentrations may cause difficulties also by clogging of the cones. Torch constructions where the diameter reduction of the capillary is nearly at the beginning of the torch (and therefore far away from the influence of the UV-radiation) improved these effects dramatically [43]. Also flow injection or microwave digestion have been proposed to eliminate this matrix problem and concomitant memory effects [23]. 3.2.2. The case o f selenium Many authors have described the peculiar behaviour of selenium in ICPMS in the presence of carbon containing solvents or compounds. Enhancement of the selenium signal has been observed in the presence of organic solvents in the liquid sample by different authors [44,45]. This sensitivity changes, well known in the Q-ICP-MS determination of Se in carbon containing matrices, have also been noticed [24] by DF-ICP-MS (see Figure 8 at mass 82). The enhancement effect of carbon can not be corrected by the use of internal
139
Double Focusing-Inductively Coupled Plasma-Mass Spectrometry
standards. It seems that the ionisation efficiency of selenium increases drastically in the presence of carbon due to charge transfer between C § and Se ~ [24]. Such matrix interferences could be compensated by using constant carbon levels in the plasma. Following this philosophy a method for the determination of Se by Q-ICP-MS in human serum has been recently published [46]. However this methodology has not been transferred to the analysis of Se in biological materials by DF-ICP-MS yet.
9
" 5-fold diluted human serum
9
9 10-fold diluted human serum
1.8 X
.~>~
9
~
-
_
_
_
.
_
_
_ _ . _
--..~ ~ =
.o ~
9 5-fold diluted human serum plus internal standa~on
AA
1.4
f2~ O
~ .~
_
,~. 1
__
.
_
.
_ _ _
. ___ A'k
9
..................................................
,,--~
,,
9149 .
.
.
.
~ .......................................
.................
9
0.6 r~
;~ -~
.
"
0.2
"-i~
9
~a'"
is
. 9
"~
.
"-
~
9
O
e,I
'q"
~4p
oO
ep
eq ,-.4
,q" ,..,
~p ,.-,
oo ~
~ e4
e4 e,I
'q' e4
Mass (amu)
Figure 8. Matrix effects due to the serum matrix [From Ref. 24, with permission] 3.3. Sensitivity and limits of detection Not only higher resolution can be achieved by resorting to double focusing. If needed, an additional advantage of DF-ICP-MS is that at R=300 it offers better limits of detection than Q-ICP-MS systems. The instrumental background at R=300 is 10 to 100 times lower than with Q-ICP-MS and therefore the instrumental limits of detection are in the pg.L "t range. It has been shown that for noble metals (in blood [35,38], urine [29,36,37,39], and tissues [47]) the limits of detection obtained with DF-ICP-MS arc up to two orders of magnitude lower than those achieved with Q-ICP-MS. The superiority of DFICP-MS for the determination of blood Pt and Au and urine Pt levels (in both unexposed and exposed population) as well as grass Pt levels in unpolluted
140
J.M. MARCHANTE-GAYON et al.
areas, has been clearly demonstrated. Adsorptive voltammetry offers comparable limits of detection, but DF-ICP-MS is faster, less sensitive to interferences, does not require total mineralisation of the sample and is capable of multi-elemental analysis [35,36,39]. Reference "values or concentrations" for environmental contaminants serve an important role in environmental health investigations and studies. They provide information about the prevalence and magnitude of exposure, which can be used as a basis for comparing concentrations in subjects who have suspected or known exposure to a given point source. To illustrate this, let us suppose that a community is located near a hazardous waste site or a plant where metals are processed. By comparing the urinary metal concentrations in people of that community with the reference values for normal people it is possible to determine if that community has had an elevated or unusual exposure. In this context, limits of detection of 1.0 ng/L and 0.85 ng/L for uranium and thorium respectively in human urine have been reported recently [48] after a simple 1:10 dilution of the samples followed by analysis using DF-ICP-MS. In this study, uranium was detectable in 96.6% of the 500 specimens (at a mean level of 11.0 ng/L) while thorium was detectable in only 39.6% (at a mean level of 1.01
ng/L). However, it should be stressed here that the limits of detection attainable by DF-ICP-MS at low resolution are so low that reagent blanks and memory effects must be drastically reduced to make full use of the possibilities of these powerful instruments. Thus, L. Moens et al [22] found that the limits of detection for Ag in serum were 10-100 times higher than the instrumental limits of detection due to the high blank values caused by memory effects. In order to minimise blank signals it is crucial to ensure minimal exogenous contamination by resorting to clean room facilities and thorough purification of all the acids by sub-boiling distillation [23,28,29,36-42]. Moreover, continuous checking for the sought element(s) in any reagent used for final analysis is mandatory. Limits of detection obtained in our laboratory [49] in five-fold diluted human serum for 14 elements at both 300 and 4000 resolving power are given in Table 4. As can be observed, most detection limits are near or below 0.1 ng/g with the exception of 27A1 and 64Zn which are limited by blanks and not by instrumental detection limits. For heavy elements, such as uranium, detection limits below pg/g levels can be obtained.
141
Double Focusing-InductivelyCoupled Plasma-Mass Spectrometry
Table 4 Detection limits obtained in the direct analysis of five-fold diluted serum samples [49] for 14 trace elements (either at R= 300 or 4000, as required) Monitored isotope 27A1 43Ca 52Cr 5SMn SaFe
59Co 63Cu 64Zn 8SRb 8SSr 98Mo
ll4Cd 2~
238U
R used 4000 4000 4000 4000 4000 4000 4000 4000 300 300 300 300 300 300
Detection limit (ng/g) 0.35 0.01 0.01 0.02 O.12 0.02 0.09 0.34 0.009 0.006 0.02 0.003 0.02 0.0005
3.4. Biomedical applications. One of the important applications of the extreme sensitivity afforded by the DF-ICP-MS instruments could be the confirmation/establishment of "reference values" for trace element content in organs and tissues of "normal" populations and using such data to be able to diagnose health and disease status, related with anomalous trace element total contents [24,25,50]. For example, DF-ICP-MS has been applied to measure serum AI basal levels in healthy people [51 ]. In order to avoid spectral interferences of the CN § ions not resolved from the A1 peak at low resolution, it is necessary to work at R=3000 (see Figure 9). There, the limits of detection for AI determination in human serum by DF-ICP-MS are one order of magnitude better than those obtained by Electrothermal Atomic Absorption Spectrometry (ETAAS) which has been for many years the preferred technique for these determinations. It appears that by minimising exogenous contamination and lowering instrumental detection limits by resorting to these of DF-ICP-MS a fin'ther lowering of "normal" or basal levels of AI in human serum from the present accepted 2 ~tg.L value to values below 0.35 ~tg.L"l is possible [51 ]. The amazing capacity of healthy kidneys to clear up aluminium from the body is even more astonishing in the light of these fmdings [51 ].
J M. MARCHANTE-GAYON et al.
142
1400
12ClSN+ 13C14N+
1200
1000
~, 8oo v
" 600
27A1+
_c 4O0
200
i
0 26.96
26.98
27.00
27.02
27.04
27.06
27.08
Mass
Figure 9. Mass spectrum of basal AI in a five-fold diluted human serum from a healthy individual showing the resolution of CN+ type spectral interferences at R=3000. [From reference 51, with permission] Single and multielemental analysis of body fluids (blood, serum, urine) using DF-ICP-MS has been undertaken by several authors [23-25,50,52] but routine measurements of large numbers of clinical samples have not been published so far in relation to "normal" or "abnormal" clinical situations, e.g. for uraemic patients [53,54]. In this vein, in our laboratory we have recently completed a study of the levels of 14 trace elements in 59 healthy subjects (41 male and 18 female, blood donors) by DF-ICP-MS and compared such levels with those found in 14 renal failure patients undergoing haemodialysis [49]. The results obtained are summarised in Table 5. As can be observed, lower values for the essential elements Fe and Zn and also for Rb and higher values for Cu, Sr, Mo, Cr and AI were detected in the haemodialysis patients. To give an insight of the ranges found, Figure 10 shows the plot of the concentrations measured for Mo and Zn for both types of sera. As can be observed, there is a clear distinction between healthy individuals and uraemic patients in their Fe and Zn content.
143
Double Focusing-Inductively Coupled Plasma-Mass Spectrometry
12.0
10.0 Uraemic patients
9
8.0 O
~-
6.0
#
g~ 0
o
4.0
Blood d o n o r s
Q~
2.0
o
o 00
.000
i
i
1
.200
.400
.600
9800
1.000
1.200
Zn concentration (pg/g)
Figure 10. Concentrations found for Mo and Zn in serum from blood donors (0) and uraemic patients (*).
3.5. Applications to food samples The analysis of trace elements in the diet is very important in order to better protect the health of the consumers. In this context, a study was undertaken by Caroli et al [55] to investigate the average levels of a number of key elements in several types of honey, with special regard to the influence of the various honey processing steps. Thus, As, Cd, Pb, Pt, Sn and V were determined by a DF-ICP-MS instrument working at R=300 while Cr, Cu, Fe and Ni were determined working at R=3000. Multielemental analysis of a wide range of elements in milk whey, human milk and infant formulae using DF-ICP-MS has been recently published by two independent groups [56,57]. In the First work, milk samples were diluted 1+4 for minor and trace elements and 1+1999 for major elements, with ultrapure water and the addition of Ga, Y, Rh, In and TI as internal standards (Table 6). In the second paper [57], milk samples were microwave digested with nitric acid and hydrogen peroxide and addition of Rh, In and Re as internal standards was used (Table 6). In both eases, DF-ICP-MS at R=3000 demonstrated that polyatomic interferences in milk whey samples were well separated, with the exception of
144
J M. MARCHANTE-GAYON et al.
Table 5 Trace and ultratrace concentrations found in blood donors and haemodialysis patients. Values are given as mean and standard deviation or concentration range when the lowest value is below the detection limit
Element
Concentration units
Ca Fe Cu Zn Sr Rb Mo Cr Mn Cd Pb U Co AI aLOD = Limit of detection
~tg/g ~tg/g l-tg/g ~g/g ng/g ng/g ng/g ng/g ng/g ng/g ng/g ng/g ng/g ng/g
Healthy people (n=59) 92.3 + 7.3 1.47 + 0.38 0.90 + 0.15 0.83 + 0.11 16.3 + 6.0 141 + 34 0.81 + 0.35 c ~ ~ '
.,1
ON'LINE'MW'D!GESTION'HGI Ar ]
~,
:,~:==3~~
phases
Double Focusing
~ HBr[~~ KBrOj
aste
Digestor
N,B~ "~,T,,I
Figure 17. Schematic diagram of the coupling MW-HG-DF-ICP-MS
5.1.4. DNA adducts quantification The main obstacle for the quantitative determination of adducts of DNA components is the impossibility of combining quantitative determinations and the elucidation of unknown structures. Known components can be determined precisely (using accelerator mass spectrometry) or newly formed unknown adducts detected (with post-labelling), but to detect and quantify unknown adducts without synthetic standards is not possible. With electrospray ionisation mass spectrometry (ESI-MS) only, the structures of unknown adducts could be elucidated, but again without pure synthetic standards quantitative results are difficult to obtain [98]. Only by combining both HPLC-ESI-MS and HPLC-ICPMS it is possible to elucidate the structure and determine the concentration of unknowns [99] and so of DNA adduct in the same sample [98]. The natm,ally common feature of all nucleotides, the phosphate group, can be employed for the purpose. The detection of phosphorus at mass 31 can be used in combination with HPLC separation of the DNA adducts using simply inorganic phosphate for quantification as the retention time for phosphate is different to that of the DNA adducts. In this context, the use of DF-ICP-MS is mandatory in order to avoid interferences from 15N160 and 14N16OIH by resorting to the use of 1500 as resolving power [98].
J.M. MARCHANTE-GAYONet al.
166
Table 11 Identification of organic solvents-induced interferences in ICP-MS.
Isotope
24Mg 25Mg 26Mg 27A1 3~p 39K 42Ca 44Ca 45Sc 5~ ' 5~ 51V 52Cr 53Cr 54Fe' 54Cr SSMn 56Fe 57Fe
A~COz
CxH~
CxHyNOz
CN CN, CHN
CH30 C3H3 C2H20
CO2 38Ar12C 4OAr12C 4~
5SNi' 5SFr
C4H2 C4H3 C4H5 C4H6 C4H7 C4H8 C4H9
59Co
6ONi 61Ni 62Ni 63Cu 64Zn 65Cu 66Zn 67Zn 68Zn 69Ga 7OZn 71Ga 72Ge 73Ge 74Se 75As 76Se' 76Ge 77Se 78Se 79Br 8OSe
CxnyOz
C2 12CI3C, C2H C2H2 C2H3
36Ar!2C160
4~
C5 CsH C5H2 C5H3 C5H4 C5H5 C5H6 C5H7 C5H8 C5H9
C6H C6H2 C6H3 C6H4 C6H5 C6H6 C6H7 C6H8
CHO2
C3HO C3H20 C3H30 C202, C3H40 C3H50 C3H60, C2H202 C3H70, C2H302 C3H402
C4HO C4H20 C4H30 C4840, C302 C4H50, C3HO2 C4H60, C3H202 C4H70, C3H302 C4H80, C3I"I402 C3H502
CsHO C5H20 C5H30 C5H40
C2NO C2H2NO
Double Focusing-InductivelyCoupled Plasma-Mass Spectrometry
S~Br 82Se 85Rb 86Sr
C6H9 C6Hlo
167
C5H50 C5H60, C4H202 C4H502 C4H602,C3H203
5.1.5. Organic solvents-induced interferences DF-ICP-MS is an effective means to elucidate the influence of organic solvents, used as mobile phases in HPLC, in ICP-MS analysis Using higher resolutions the analytical determinations derived from low resolution measurements can be checked for accuracy. In Table 11 most of the polyatomic interferences observed and identified by precise mass measurement are compiled [91]. All of them may impede application of reversed phase chromatography or introduce problems for the direct investigation of organic matrices with quadrupole-based instrumentation. However, most of these interferences could be overcome by using higher mass resolution settings in a DF-ICP-MS instrument. In the mass interval 24-86 amu the possible appearing polyatomic ions in the presence of organic solvents can be sub-divided into four groups: (i) ArCOz with z=0 or 1, (ii) CxHy with x = 1-6 and y--0-10, (iii) CxHyOz with x = 1 or 2, y=08 and z=l-3 and (iv) CxNHyOz with x =1 or 2, y=0-2 and z-0 or 1. As it can be seen, these interferences continuously cover the whole interval 24-86 amu, and some of them may appear even above 100 amu. Their distribution depends strongly on the operation conditions chosen, such as generator power, nebuliser flow rate, sample uptake rate and organic solvent concentration, and also on the instrument itself, the sample introduction system used and its operational conditions [91 ].
5.2. Gas chromatography (GC) In comparison with HPLC-ICP-MS, GC-ICP-MS offers a higher chromatographic resolving power and 100% sample introduction efficiency (higher sensitivities); it allows a more stable plasma and gives origin to fewer spectral interferences as a result of the plasma being dry; moreover, this coupling leads to less sampling cone and skimmer wear. Of course, GC-ICP-MS can only be used for the separation and detection of volatile and thermally stable compounds or compounds that can be derivatised into a volatile form. Unfortunately, no applications of its use with DF-ICP-MS have been reported so far. Also the coupling GC-ICP-MS is somewhat more complicated as a heated transfer line is usually required, such that condensation of the species and, hence, peak broadening can be avoided. In the last years a new interface for the coupling of GC to the ICP-MS has been described [ 100] which does not require
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the heating of the transfer tube between the gas chromatograph and the ICP-MS simplifying the coupling and decoupling of both instruments [100]. The coupling of GC to a DF-ICP-MS instnnnent is under way in our laboratory for the speciation of sulphur compounds in saliva and its relation to bad breath. For example, Figure 18 shows the separation of 8 volatile sulphur compounds by GC as detected by DF-ICP-MS at mass 32 at R=3000 to eliminate the interference from 1602+. In this case the injection was 1 ml of air containing about 200 ng of each compound. In the same figure the chromatogram obtained for a sample of saliva fermented anaerobically for 24 hours is presented. As can be observed, many sulphur compounds can be SH 2
MeSH EtSH
MeS-SMe I MeSMe MeSEt
EtSE t
l
.1 Standard
Sample
Figure 18. Separation by GC and detection of volatile sulphur compounds at mass 32 using DF-ICP-MS at resolution 3000.
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detected in the real sample and some of them can be readily identified by their retention time. Unfortunately, many other compounds remain unidentified so far. This is an unexploited area worth of future research.
5.3. Capillary Electrophoresis (CE) In principle, the field of application of CE is similar to that of HPLC, but CE has several advantages such us higher efficiency, small sample volume requirement, shorter analysis time, minimal buffer consumption and higher sample throughput. In addition, CE is especially suitable for the separation of biological macromolecules, e.g. proteins. The major drawback in the application of CE to the speciation of real samples is the small sample volume (up to 50 nL) which requires a very sensitive detector to match the (low) naturally occurring analyte concentration levels. The critical need for sensitivity of the specific detector coupled for speciation points to DF-ICP-MS, working at low resolution, as the preferred choice [101 ]. The key to the successful realisation of a coupled system for trace metal speciation is the design of the interface. This is especially true for CE-ICP-MS [102]. Whereas HPCL-ICP-MS coupling is relatively easy to achieve (flow rates in HPLC fit well with the nebulisation flow rate in ICP-MS) for a CE-ICP-MS interface we have to provide (a) an electrical connection; (b) to adapt the flow rate in the capillary with the flow rate of the nebuliser, to prevent suction effects in the nebuliser ruining the separation and (c) preserve a good sample transport (for sensitivity). The analytical characterisation of a CE-DF-ICP-MS system for the separation and determination of three chemical species of Hg (CH3CH2Hg +, CH3Hg+ and Hg 2+) has been recently applied in our laboratory [103]. The three Hg compounds were separated as mercury-cysteine complexes by CE at 20 kV using a 20 mM sodium tetrabomte buffer (pH--9.3). In this interface, the capillary was inserted through a T-piece into the Meinhard nebuliser and held in place, after optimisation of its position, with a ferrule fitting. Grounding of the capillary was achieved by the use of a coaxial '~nake-up liquid" flow which was mixed with the CE effluent prior to nebulisation. This solution was pumped through the vertical arm of the T-piece via a teflon tube using a peristaltic pump. To complete the electrical circuit, a cathodic connection was made through the same ann of the T-piece by placing a platinum wire between the teflon tubing and the ferrule[ 104]. The self-aspirating nature of the commercial concentric nebulisers induce a laminar flow (suction) in the capillary which can ruin the performance of the CE separation. Thus, the aspiration rate in the nebuliser, strongly influenced by the nebuliser gas flow rate, is a critical parameter. The optimum nebuliser gas
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flow rate will depend upon a combination of its effect on the electrophoretic migration time and resolution and on the ICP-MS signal intensity. If the nebuliser gas flow is too low, the generation and transport efficiency of a f'me aerosol into the ICP will be compromised. The concentration detection limits observed are compiled in Table 12 [103]. From this Table, it is apparent that the detection limits obtained using conventional UV absorption are significantly higher than using ICP-MS detection. Moreover, as expected, the use of a DF-ICP-MS detector (working at resolution 300) gave improved detection limits in all cases as compared with quadrupole-based ICP-MS intruments. Table 12 also shows that the "sample stacking injection" [103] can be used to improve the detectability in the CE separation and f'mal determination of mercury compounds. Much more work is needed to render CE-ICP-MS techniques a real competitor in trace element speciation of HPLC-ICP-MS. Table 12 Detection limits (~tg/L) for CE-UV, CE-Q-ICP-MS and CE-DF-ICP-MS for mercury species[ 103] Method CE-U:v (injection volume = 0.163 ~tL) CE-Q-ICP-MS (injection volume = 0.350 ~tL) CE-DF-ICP-MS (injection volume = 0.450 laL) SS-CE-DF-ICP-MS (injection volume of 3 ~tL with sample stacking)
Hg2§ 500 81 25 4
CH3Hg+ CH3CH2Hg+ 680 750 128 275 54 84 7
5.4. Off-line strategies The capability of flatbed electrophoresis combined with DF-ICP-MS has been reported by Comelis et al [105]. Rabbit serum samples were separated by isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Rabbits received intraperitoneal injections of Ga (III) and In (III) in physiological buffer. Each rabbit serum sample was rtm twofold and, aider separation, one gel was silver stained (in order to detect and identify serum proteins) and the other replicate was cut into segments, which were digested with aqua regia and measured for Ga and In at R=7500 (in order to avoid spectral interferences). Using such strategy, it was observed that both elements are bound exclusively to transferrin. Similar strategy has been used by the same group [106,107] in order to develop native two-dimensional electrophoresis methods for the separation and detection of platinum carrying serum proteins. In the first dimension IEF was
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performed using immobilised pH gradients (IPGs), while PAGE was done in the second dimension. Detection of proteins was achieved again via silverstaining. For the DF-ICP-MS determination of platinum, one gel was cut into small pieces and the element extracted with aqua regia. 5.5. Future of DF-ICP-MS for speciation It should be pointed out that the main intrinsic disadvantage of DF-ICPMS for speciation of biological materials is, as for other elemental detectors, its atomic character which prevents any molecular information to be obtained. Its coupling with a powerful separation technique (chromatography or capillary electrophoresis) alleviates this limitation via hybrid techniques. However, identification of the metal binding biomolecules using such techniques, in real samples relies only in the observed retention time. Such parameter is not enough because it depends on the chromatographic conditions and, what is more, an adequate standard (appearing at the same retention time in the same conditions) should be available for final identification of a given biometallic compound in the samples. While this is not usually a difficult problem in trace element speciation of organometallic compounds in the environment (because we know virtually all metal species concerned) it can be a terrible headache in biological systems where the possible metal-biomolecules forms are unknown [93]. In the organisms, the sought metal or semimetal has been usually integrated in the biological material by the living organism, transformed into often tmknown compounds and so buried into a very complex matrix. Awareness of the need for complementary techniques providing matching results [82] for such problems is important at this stage. We are needing, more and more, reliable tools for identification and confirmation of metal-biocompounds before going into the mandatory last step of quantification of the sought species in a given biological sample. For identification purposes, the use of retention times provided by a single chromatography-detector system is not enough; different-principle-based separations with different detectors for the same problem should give matching results [87] and, therefore, could constitute a helpful approach to solve the speciation problem. Confirmation techniques and methods are eventually required to be sure that the separated species are the compounds we expected (and just a pure compound). Isolation, purification, preconcentration, etc. of the unknown "pure" species is advisable before final characterisation and confirmation of its nature/purity by molecular techniques (preferably common organic mass spectrometry tools, NMR etc.).
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It is opportune in this paper to point out that "high resolution" achieved in the DF-ICP-MS instrument refers only to isotopic masses considered (it is atomic). Of course, in speciation different molecular species of the same element not separated adequately in the chromatographic column will coelute and our "high resolution" setting in the atomic detector will be useless. All the above considerations matter in the search for quantification strategies and final validation of new speciation methods developed. Adequate quantification of the species requires first a "neat" compound separation, then a sensitive and element-specific detection and finally a validation of the determination method developed. In the search for validating strategies we can resort to the use of Certified Reference Material (CRMs) for a given species. These "speciated" CRMs are unfortunately difficult to produce [108] and scarce today. An alternative strategy for validation is the use of alternative well established ("reference") methods producing statistically identical results. It seems that this second approach is more flexible and can be advantageously used in many laboratories lacking the CRMs for validation. In this vein, we believe that such highly qualified "reference" methods for speciation results validation could be provided by Isotope Dilution ICP-MS, considered as definitive or "primary" measurement methods [109]. The application of postcolumn isotope enriched spikes or of synthesised isotope enriched species, as described by Heumann [110], are approaches of a great potential for accurate quantification of metalbiomolecules in biological systems [82]. In this vein, the ability for high precision isotope ratio measurements afforded by DF-ICP-MS along with its extreme sensitivity at R=300 warrant a brilliant future for this detector, particularly in trace element speciation of biological materials. REFERENCES ~
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Chapter4
Field-flow fractionation-inductively spectrometry
coupled
plasma-mass
Ramon M. Barnes, and Atitaya Siripinyanond Department of Chemistry, Lederle Graduate Research Towers, University of Massachusetts, 710 N. Pleasant Street, Amherst, Massachusetts 01003-9336, USA, and University Research Institute for Analytical Chemistry, 85 N. Whitney Street, Amherst, Massachuetts 01002-1869 USA I. INTRODUCTION In the elemental analysis of biological, biomedical, environmental, geological and other natural materials, three key objectives are often sought: (1) identification and quantification of elemental concentrations, (2) identification and quantification of compounds containing metals, and (3) evaluation of bioavailability and health effects, mobility, transport, fate assessment, and toxicity [1,2]. Nowadays, information on elemental concentration is insufficient for these objectives owing to the increasing knowledge of metabolism and biological effects of elements. The determination of chemical species is essential for characterizing the biogeochemical cycle and contaminant transport in terrestrial and aquatic ecosystems, and assessing their risk to biota and humans [3]. The primary goals in many elemental analysis of biological and environmental substances are identification and quantification of their chemical forms. "Elemental speciation" underpins elemental bioavailability, in vivo absorption and distribution, mobility and toxicity studies. Over the past 2 decades, various definitions of speciation have been proposed. This ambiguity was officially validated by the International Union for Pure and Applied Chemistry (IUPAC) definition which denotes speciation as "the process yielding evidence of the atomic and molecular form of an analyte" [4]. The term "speciation" has been used in different ways, however. Only recently, three IUPAC Divisions (represented by the Commission on Microchemical Techniques and Trace Analysis, the Commission on Fundamental Environmental Chemistry, and the Commission on Toxicology) attempted to provide clear definitions of concepts related to speciation of elements. Speciation of an element has been defined as "distribution of an element
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amongst defined chemical species in a system", whereas ffactionation has been defined as "process of classification of an analyte or a group of analytes from a certain sample according to physical (e.g., size, solubility) or chemical (e.g., bonding, reactivity) properties [5]. Challenges and trends of analytical speciation in biological, environmental, and other natural systems have been surveyed [4,6-12]. Elemental speciation is otten achieved by combining two complementary techniques since a single one is often insufficiently selective or sensitive. One provides an efficient and reliable separation procedure, whereas the other provides adequate detection and quantification. Ideally, technique coupling requires sample introduction compatibility. Minor instrumental modifications with maximum interface efficiency and response of each technique is desirable. In addition, real-time data acquisition for the separation process is expected. Separations include both chromatographic and nonchromatographic techniques. In elemental analysis, inductively coupled plasma (ICP), microwave induced plasma (MIP), and glow discharge (GD) sourceatomic emission and mass spectrometry (ICP-AES, ICP-MS, MIP-AES, MIPMS, GD-AES, GD-MS) are of primary importance, owing to their multielement detection capabilities [13,14]. On-line coupling between separation procedures and ICPs have been assessed and occasionally reviewed [15-18]. Commonly, the analytical technique selection criteria used for elemental speciation depend primarily on the study objective. For most biomedical and environmemal samples, reversed-phase (RP), ion-exchange (IE), size exclusion chromatography (SEC), and electrophoresis are preferred liquid separation methods. The three former are chromatographic techniques that typically provide moderate resolution. Became their mobile phase flow rates are wellsuited to ICP-MS sample introduction flow rate (e.g., ~0.1-2 ml min-~), coupling between these chromatographic techniques with ICP-MS is physically straightforward. The methods are subject to artifacts, however [2]. In contrast, capillary electrophoresis (CE) is a popular high-resolution, nonchromatographic technique for analytical separation [19]. Despite its high resolution, CE operates at low flow rates (e.g., nl min'~), which sometimes leads to unstable ICP-MS operation. An appropriate interface design is required to provide an electrical connection, to adapt the electro-osmotic flow rate in CE to the sample introduction flow rate of ICP-MS by using a make-up solvent and to prevent a suction effect between the nebulizer and the CE capillary [20]. The combined CE-ICP-MS technique has been spearheaded by Tomlinson et al. [21], Olesik et al. [22], and Lu et al. [23]. Following these three pioneered articles, CE-ICP-MS applications have developed. Other interface configurations have been evaluated [24] and commercial interfaces and nebulizers are now available [25] (Meinhard | SB-30-A3 and DIHEN nebulizer
Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry
181
for CE (J.E. Meinhard Associates, Inc., Santa Ana, CA, USA),Microneb 2000 and MCN 1000 microconcentric nebulizers (CETAC Technologies, Omaha, NE, USA), and a MicroMist AR30-1-F02 nebulizer (Glass Expansion Pty Ltd., Australia) ). A modified microconcentric nebulizer MCN 100 developed by Prange and SchaumlOffel was reported and commercialized recently [20,26]. Majidi et al. demonstrated an approach to improve analytical sensitivity and detection limits using multicapillary parallel separation interfacing to ICP-MS [27]. The multicapillary comprises several individual capillaries with identical internal diameters and lengths placed in a loose bundle. An increase in signal intensities proportional to the number of capillaries was observed. Instnarnentation development and applications have been reported continuously [28-34]. For interested readers a series of contemporary papers by Michalke et al. is recommended [35-37]. In their on-line CE-ICP-MS, a laboratory modified commercial concentric glass nebulizer initially employed with a cyclonic spray chamber was fabricated [35-37]. However, as with SEC, artifacts from instrumental, column or packing materials, in CE might limit trace and ultra-trace metal quantitative analysis for complex polymer systems, colloids or biomolecules. Therefore, as predicted by Barnes [1,2] an alternative separation approach, like field-flow l~actionation (FFF), for elemental speciation in biomedical, biogeochemical, and natural samples might provide less artifactdependent separations. Field-flow fractionation is a non-chromatographic elution-based separation technique that is capable of separating and characterizing materials in the macromolecular and colloidal size range and larger. Since ICP-MS is a rapid and sensitive elemental detection technique, the combination of FFF with ICP-MS should provide detailed elemental characterization of macromolecules and particles. Field-flow fractionation-ICP-MS was first reported in 1991 [38], and a number of publications has appeared since [39-42]. In these papers natural suspended particulate matter, clay minerals, and soil were analyzed. In contrast, many FFF applications with conventional detectors (i.e., spectrophotometry, light scattering, etc.) have examined biological macromolecules [43-49]. Now FFF is a demonstrated practical on-line separation tool for ICP-MS elemental detection. However, FFF-ICP-MS for biomedical research and clinical analyses is still in its infancy and unexplored. Only a single report demonstrating the feasibility of linking F1FFF with ICPMS for analysis of several protein standards has appeared [50]. As FFF provides gentle separation with delicate and shear-labile species with minimal loss of biological activity, FFF-ICP-MS should give unique separations and characterization of natural biopolymers (e.g., humic acid, hyaluronic acid). Therefore, the FFF-ICP-MS technique is worth examining as a complementary
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BARNES and SIRIPINYANOND
tool for elemental speciation. Considering the increasing number of publications and presentations made at several international conferences, the exciting FFF-ICP-MS journey has begun. In this chapter, the basic FFF principles and a general overview of FFF techniques are summarized. Different FFF modes are described briefly. Selected applications to environmental and biological samples are reviewed, and specific FFF-ICP-MS applications are highlighted. Recent experimental results obtained from our research laboratory are presented as possible applications of the technique for biological and environmental analysis. A forecast of the future direction of FFF-ICP-MS is also presented. 2. GENERAL OVERVIEW
The FFF concept was first envisioned and documented in 1966 as a threepage short communication by the late Professor J. Calvin Giddings[51]. The FFF idea emerged late in 1965 during his vacation in Wyoming, where he spent a night in a motel in Evanston, a cowboy town [52]. A banging radiator kept him awake, so that he started thinking about the diffusion problem [53]. He imagined using some kind of force field to restrain a mixture within a narrow layer while differential diffusion acted to allow some species to escape further from the layer than others [52]. Thermal diffusion was the first appropriate field that came to mind. Then, the idea was first implemented experimentally at the University of Utah, Chemistry Department. For the interested readers brief accounts of the early FFF history are given by Giddings et al. in a worthwhile paper, published in 1981 [52]. Field-flow fractionation is a versatile technique capable of separating and characterizing materials with an application range from less than 103 daltons (Da or g mol l) to particle diameters of 100 pan [53]. Similar to chromatography, FFF is an elution-based technique. The fractionated components can be detected on-line and/or collected off-line. Unlike chromatography, however, FFF is achieved in a thin ribbon-like, open channel with no stationary or liquid phase. By avoiding a stationary phase, retention is not induced by a distribution between two or more phases, and FFF is not a chromatographic method [54]. The essence of FFF is to apply a force perpendicular to the channel flow stream (Figure 1). This force drives molecules toward an accumulation wall where they encounter slow-moving laminar fluid streamlines. Different size sample components are driven different average positions in the channel flow, causing them to migrate at a characteristic rate. Thus each component leaves the channel at a different time related to its molecular weight. Fundamentally, these primary driving forces
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Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry
can be generated by a number of fields or gradients. The field is applied to compress sample components against an accumulation wall while the channel flow is stopped temporarily, immediately after sample is introduced. This results in numerous FFF sub-techniques that are generally applicable to different materials (Table 1). Table 1 Applicable range of particle diameters by FFF techniques FFFsubtechnique Sedimentation Thermal Flow Electrical
Applicable particle size range (nm) 100-100,000 2-100 1-100,000 40-1,000
Applicable MW range ..(g mo1-1, Da). 106-10 ~5 104-10s 103-1015 102-106
Four retarding fields have been widely studied. The most common is sedimentation [55], usually generated in a centrifugal force but sometimes by gravity [56]. The centrifugal or gravity force acts perpendicularly to the flowseparation axis. This sub-technique is known as "sedimentation FFF (SdFFF)". Other important fields include a temperature gradient (thermal FFF, ThFFF), in which thermal diffusion is the separation driving force [57]; electrical field (electrical FFF, EIFFF), in which force depends on particle charge [58]; and a cross-flow stream of carder liquid (flow FFF, F1FFF) [59], in which the force originates in the friction of the cross-flow stream moving across the components. Among them, F1FFF is most universal, because of its wide applicable range, as illustrated in Table 1. Broad range of FFF applications in the Giddings' research group are listed in Table 2 to illustrate the scope. Since the first paper appeared in 1966, published results have continually grown (Figure 2). Thus far, eight international FFF symposia have been held. The most recent one was held in 1999 in Paris, France. The next will be held in Golden, Colorado, June 2001. The general principle of FFF techniques is given in many reviews [43,61-69] and textbooks [70-72]. Several useful FFF related web sites are also available. Among them, the FFF webspace "http://dns.unife.it/~rskf' maintained by Pierluigi Reschiglian is very web page "http://www.rohmhaas. com/fl~' by Mark R. Schure at Rohm & Haas, where all the publications of FFF and related techniques are compiled and listed for
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BARNES and SIRIPINYANOND
Channel inflow
Outflow ,
AppliedS
side view
o"
... \
Parabolic flow profile Flow r----
.s
.%
/
Inflow and sample injection
Outflow (to detector)
_.
top view Z
Y
X
Figure 1. Principle o f FFF separation X are the smallest sample particles, having highest diffusion coefficient Y are the moderate size sample particles, having moderate diffusion coefficient Z are the largest sample particles, having lowest diffusion coefficient ly is a mean sample layer thickness of distance of a cloud of particles Y Briefly, separations are performed within a flat open channel with a rectangular cross-section and triangular end pieces where the sample and cartier liquid enters and leaves. Fractionation is achieved by the balance between the field force applied on sample particles toward the accumulation wall and the diffusivity of sample particles acting against the field force. The field force pushes X, Y and Z into thin clouds or layers of different mean thickness. Owing to the highest diffusivity of sample X, a mean sample layer of particles X is thicker than that of Y and Z. Therefore, a cloud of particles X meets the higher speed region of parabolic profile, thus particles X elute earlier than particles Y and Z.
Field-FlowFractionation-InductivelyCoupledPlasma-MassSpectrometry
185
each year, is convenient. A discussion forum "the FFF mailing list" maintained by Frank von der Kammer, is where questions and announcements related to FFF are posted. To register, a requested message can be submitted to "
[email protected]. de". Table 2 Some particles characterized by FFF methods in the FFF Research Center (FFFRC, University of Utah)and FFFractionation Laboratories (listed in 1990,
[60]) Category Samples Samples Polystyrene Inorganic Selenium Polyvinylchloride Nickel Polybutadiene Glass Polyurethane Silica Hematite Polymethylmethacrylate Styrene-butadiene CLay Grafted polybutadiene-PMMA Limestone Vinyltoluene t-butadiene Biological Red blood cells White blood cells Epoxy-acrylic resin Alkyl resin Human lens particles Teflon Albumin particles Emulsions Soybeanoil Casein particles Safflower oil Viruses Perfluorocarbon Pollen grains Liquid crystal Mitochondria Liposomes Lysozomes Environmental Fly ash Coal liquefaction residue Ground coal Waterborne colloids Reprinted from [60] with kind permission of the American Chemical Society.
Category Polymer
Presentations made in 1996 to 1999 from the University of Ferrara (Italy) research group are available to download on line. The web site "http://www.wsc.monash.edu.au/t~' of Ronald Beckett at the Water Studies Center, University of Monash in Australia also is instructive. Only a few FFF manufacturers exist with many distributors worldwide. These manufacturers include (1) FFFractionation, Salt Lake City, Utah [http ://www. fffract, com] (2) Consenxus, Ober-Hilbersheim, Germany
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BARNES and SIRIPINYANOND
[http://www.consenxus.de], and (3) Postnova Analytics, Munich, Germany [http://www.postnova.com/ueberuns/index.htm].
.~ ..~ =u = t. o -=
110 100 90 80 70 60 50 40 30
f ra U T A H
[] not U T A H I
20 =
10 0 ~
t -~
t ~-
11 .o00=0=H0 tHII
~--
t ~-
t~
O0
O0
O0
O0
O0
~
~
~
~
~
Year
Figure 2. Number of FFF papers. Source: Field-Flow Fractionation web site updated on August 22, 2000 and Web of Science updated on September 12, 2000. Note: Number of FFF publications are striking in 1997. This is probably due to many special issues in scientific journals dedicated to the death of Professor d. Calvin Giddings in 1996. 2.1 F F F M o d e s
For a given field type three basic operation forms arising from different kinds of opposing forces extend versatility and FFF variety. These are normal, steric [73], and liit-hypedayer FFF [74,75] (Figure 3). In normal mode FFF, the center of gravity of the solute zone lies very near the wall, usually extending only a few micrometers from the wall. As the 2-dimensional flow profile is parabolic, flow velocities vary fi'om zero at the walls to a maximum in the channel center. For parabolic flow the velocity at the channel center is twice the average flow velocity. The smaller particle zones extend a greater distance, compared to the bigger particles, into the channel owing to their weaker field interaction and higher diffusivity. Consequently, the smaller particles elute earlier than the bigger ones. However, as particle size increases to a critical value, where the particle cannot intercept flow lines closer to the wall than its radius, diffusion from the wall is strongly suppressed, and it no longer plays a major role in retention. This operation mode is called "steric FFF" [73]. Larger particles occupy an elevated position from the accumulation wall than the smaller counterparts. Therefore, the large particles are swept downstream and leave the channel more rapidly, resulting in elution order trend opposite to that observed for the normal FFF. As the average velocity of the carder in the
Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry
187
channel increases, particles that were in the steric region begin to experience hydrodynamic lift forces that drive them away from the position at or near the wall. This operation mode is called "hyperlayer FFF", in which the particles gain a significant elevation above the wall where they form hyperlayers [72]. The word "hyperlayer" describes particles forming thin layers above the accumulation wall, where "hyper" signifies over or above [72]. The hyperlayer mode elution trend is similar to the steric mode. A. Normal FFF |
Ill
h.~
II
I
R~61
W
B. Steric FFF i
,,,
-
y
W
...._...__.~
a
. . . . . .
C. Hyperlayer FFF ~_
II
I
I
....
e~6Xeq ,,,
'
"
~~~P~X~
~
W lff
" '
Figure 3. Schematic representation of the three major modes (or mechanisms) for FFF separations: (a) normal, (b) steric, and (c) hyperlayer. R is retention ratio, I is mean cloud thickness, Wis channel thickness y is an empirical correction factor which takes into account several non-ideal phenomena a is the particle radius (assumingthat a >>/) Xeq is a distance of a cloud of particles. It depends on the balance of the force generated by the external field and the imperfectly characterized hydrodynamic lift force.
2.2 F F F Sub-techniques
Any field or gradiem capable of providing differential displacement can be used in FFF. Each field impresses its unique selectivity dependence on particle
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BARNES and SIRIPINYANOND
properties. Relationships between force and physicochemical properties, as summarized by Giddings [76], are indicated in Table 3. Instnunental setup and principles o f each sub-technique are described in this section. Table 3 Force equations and physicochemical properties
Force equations J Sedimentafio nFFF (SdFFF) Force = m'G = m(Ap/pp)G = VpApG d~ /6)ApG Thermal FFF (ThFFF) force = DTf(dT/dx) = a k(dT/dx) = kT(Dr/D)(dT/dx)
Electrical FFF (EIFFF) force = qE
=0rE
Properties measurable from FFF retention ,
m' = m = Vp = d =
effective mass (g), G = acceleration mass, pp density, Ap = density difference (gcm -3) volume (cm3) equivalent volume spherical diameter (cm) =
DT = thermal diffusion coefficient, f = friction coefficient ~t = thermal diffusion factor, dT/dx = temperature gradient D = ordinary diffusion coefficient, T = temperature (K) k = Boltzmann constant (1.38 x 1016 gcm2 s'2Kq)
q = effective charge, E = electrical field intensity la = electrophoretic mobility, f = friction coefficient
Flow FFF (FIFFF) force = fU f = friction coefficient, U = cross-flow velocity (cm sq) = 3~ rldhU dh = hydrodynamic diameter,rI = cartier viscosity (g crn"lsq) = (kT/D)U D = ordinary diffusion coefficient (cm2 s-1) Reprinted from [76] with kind permission of the American Chemical Society
2,2,1 Sedimentation FFF (SdFFF) Sedimentation FFF is the most common FFF technique. In principle separation is caused by using either gravitational or centrifugal field forces on the particles suspended in a carder liquid (Figure 4). The gravitational or centrifugal force causes sedimentation o f the separated colloidal sample components according to the product o f their effective volume and density difference between the suspended particles and carder liquid. Like other FFF techniques, separations are performed within a flat open channel with a rectangular cross-section and triangular end pieces where the sample and carder liquid enters and leaves (Figure 1). Sedimentation FFF was first envisioned by Giddings [51] and was fn~t theoretically proposed [77] and experimentally verified by Berg et al. in 1967 [78]. A centrifugal device that bypassed the need
189
Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry
for complicated rotor seals was employed. The channel for SdFFF is usually wraped around the inside circumference of a centrifuge rotor basket. Special seals are used to close the inlet from the outlet streams and thus to prevent leakage. The channel assembly can therefore be spun at different rotation rates to control retention in the fractionation system [79]. The separation time sequence of SdFFF is illustrated in Figure 4. The retention parameter depends on the effective mass of the fractionated species and operating parameters. Despite its high resolution, the centrifugal force is too weak to induce retention of small particles (less than 10 to 30 nm). At the highest spin rates available (2500 rpm) some retention begins to occur at a molecular weight of about 106 g mol 1. Beyond this transition value, SdFFF becomes a highly selective technique. Determination of size and density of the separated particles is possible by performing the fractionations in carrier liquids of various densities.
A)
x
i
190
BARNES and SIRIPINYANOND
Pump end B A
B.
\ --
~--
-i chmnnel
to
t9
:
u
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_
!
_~
I
Retention time tR (minutes)
I Time sequence
a
b
c
8all!pie injection end
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84nell sampte component eiutod
relaxation-no
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d
Large ~ cmaponent eluted
Figure 4. Schematics of SdFFF A) principle of SdFFF Three particle sizes are shown: smaller particles (X), bigger particles (Y), and floating particle (Z) with a density smaller than that of the solute. (Reproduced from [43] with kind permission of the American Association for the Advancement of Science) B) time sequence of separation using SdFFF (or SFFF): a) sample injection and relaxation before flow; b) sample separation by flow; c) elution of smaller particles; d) elution of larger particles. (Reproduced from [80] with kind permission of the American Association for the Advancement of Science)
2.2. 2 Thermal FFF (ThFFF) In the FFF family ThFFF was the first techniqueimplemented [81,82]. A temperature gradient is the separation driving force in ThFFF (Figure 5). A thin ribbon-like channel is slotted in between two metallic blocks with high heat conductivity such as highly polished copper bars with coated nickel or chromium surfaces (Figure 5). Generally, the copper bars act as the heat transfer source to maintain the temperature gradient between the top (hot) and the bottom *(cold) walls. Several holes are drilled in both copper block sides,
Field-Flow Fractionation-InductivelyCoupled Plasma-Mass Spectrometry
191
where thermistors or thermocouples are located to control and regulate the block temperatures and the temperature gradient between the two main channel walls. Electrical cartridge heaters and carder stream inlet and outlet tubes are positioned in the top bar. The lower or cold wall is cooled by flowing a coolant through a heat exchanger unit. A channel is properly cut from a spacer comprising a low thermal conductive material (Mylar| or Teflon| and is placed between the metallic blocks. The tThFFF channel is represemed in Figure 5. Because of thermal diffusion, the separated sample componems migrate toward the cold wall. This well-known phenomenon is called the Ludwig-Soret effect. In IhFFF, unlike other FFF methods, the flow profile is perturbed by the change in solvent viscosity with position in the channel owing to the temperature variation across the channel. Theoretically, ThFFF is the most complicated FFF technique, because of the numerous assumptions and approximations. In practice, retention is linearly related to the temperature difference between the cold and hot walls. This temperature gradiem pushes particles or macromolecules toward the accumulation (cold) wall. Generally, larger particles are driven closer to the accumulation wall than the smaller species. Experimemally, the retention yields the Soret coefficiem, which is the ratio between thermal diffusion and diffusion coefficients. Therefore, thermal diffusion coetticiem can be calculated when the normal diffusion coefficient is determine experimentally one parameter when the other is known independently.
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BARNES and SIRIPINYANOND
Figure 5. ThFFF A) channel arrangement (Reproduced from [83] with kind permission of the Wiley) B) basic principle (Reproduced from [84] with kind permission of the Dekker) 11is a mean layerthicknessof low molecularweightfraction 12is a mean layerthicknessof high molecularweightfraction 2,2. 3 Electrical FFF (EIFFF) Although the system setup is seemingly simple (Figure 6), E1FFF is very difficult to implement in practice. Experimental difficulties are encountered, leading to less development of this than the other FFF techniques. Electrical FFF uses an electric field to establish a potential drop across the channel to generate a lateral flux of charged macromolecules or particles [85]. Semipermeable membranes are usually placed inside the channel to allow small ions to flow from the channel. According to Caldwell, one important consideration with early EIFFF channel construction was a gradual droop at the membrane walls [51]. Additionally, small fluctuations in the channel flow cause a wall deformation, which affected both retention and zone spreading [86]. A system was redesigned with membranes cast directly onto a porous rigid polymeric flit support [87]. This design provided a stable channel geometry, yet the electrical resistance was too high causing undesirable heat effects. Later an experimental setup was constructed (Figure 6) in which main channel parts comprise two Plexiglass blocks with chambers that enable flow through buffer solution. Two semipermeable flexible membranes were placed inside the channel to allow small ions passage and separate the channel volume from electrode compartments in the blocks. The two membranes are separated by the channel spacer material. The entire system is clamped and bolted together.
Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry
193
In the previous design a spacer that determines the channel thickness was 0.356 mm thick [88]. Platinum wire electrodes were placed above and below the spacer and were positioned 51 mm apart. Owing to the thinness of the channel compared to the spacing between the two electrodes, only about 0.356/51 or 0.7% of the applied potential was used for the separation. Later a design was constructed by Caldwell and Gao [89] in which two graphite plates served the dual role of electrical field source and channel wall. A Teflon | spacer is inserted between these plates. Since the channel walls are made of graphite, an upper limit to the practical separation fields exists owing to the electrolytic breakdown of water. Despite this limitation, the voltage generated from a very narrow gap is very high. Typically, the EIFFF channel dimensions resemble those of F1FFF. In E1FFF an external electric field is applied between the two channel walls to force charged analytes to migrate toward the wall of opposite charge. Electrical FFF exploits the differences in the electrophoretic mobility of particles to separate them. This electrophoretic mobility fundamentally depends on particle size, shape, surface charge density, and solution ionic strength. Usually, the apparent retention time is used to determine experimentally one parameter when the other is known independently
A)
PL,ATINUM W I R E
VOLTAGE
I
"
E T ~ W
N TUBE~G R SPACER
IGLASS
PLATINUM W I R E
194
BARNES and SIRIPINYANOND
Polarity
B)
+ i
i
v
i
"
-
----
t
\
r
/ coneons~ts
Figure 6. EIFFF A) instrumental setup (Reproduced from [83] with kind permission of the American Association for the Advancement of Science) B) separation principle (Reproduced from [90] with kind permission of the Elsevier) l is a mean layer thickness of particle clouds A is big particles with high charge density B is small particles with low charge density E is field strength
2.2. 4 Flow FFF (FIFFF) Similar to other FFF techniques, F1FFF separation is induced by an external flow field perpendicular to the separation axis (Figure 7). The field subsequently causes components to migrate to the accumulation wall. The physical fluid cross-flow drives all entrained particles and molecules toward the accumulation wall. This makes F1FFF an almost universal FFF technique [91]. Owing to its universality, F1FFF has been exploited as the separation method combined with ICP-MS for elemental speciation. Therefore, mathematical equations related to fi'actionation using F1FFF are given below. Flow FFF theory has been extensively described [59,60,64]. Fractionation in the FIFFF channel is achieved according to the fractionated components diffusion coefficients and hence their molecular weights. In a F1FFF apparatus, a ribbonlike channel is generally cut from a thin plastic spacer (Figure 7). A membrane having a specific molecular weight cutoff is inserted inside the FFF channel. Generally, requirements for the membrane include the following: fiat surface, rigid support, uniform porosity, suitable pore size, inertness to the carder liquids and samples, and suitable back pressure to maintain a uniform crossflow. A channel flow is introduced at one end of the channel where a smallvolume sample is injected. Practically, the channel flow is stopped momentarily alter the sample has entered the channel to allow the sample components to accumulate on the membrane, relax, and reach equilibrium
Field-Flow Fractionation-lnductively Coupled Plasma-Mass Spectrometry
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distributions. A second stream of liquid, applied perpendicular to the channel, is a cross-flow and serves as the driving force to displace particles across the thin channel toward the membrane. The cross-flow enters the channel by passing uniformly through a porous ceramic flit and then the membrane. This secondary flow is introduced to retard the movement of sample particles in the parabolic channel flow stream. This retardation provides the fractionation between particle sizes, primarily based on their diffusion coefficients from the accumulation membrane (D, cm 2 sl). The F1FFF principle is illustrated in Figure 7. Theoretically, the diffusion coefficient is related to the Stokes (hydrodynamic) diameter (d~, cm) of the component by the Stokes-Einstein relationship [47] D = kT/3xrld~
(1)
where k is Boltzmann's constant (1.38 x 1016 gcm 2 s2Kl), T is the temperature (K), and r/is the viscosity of the cartier liquid (g em'ls-l). For random coil maeromoleeules, d~ is related to molecular weight M (Da) [47] by a, = a 7v/'
(2)
where the constant A" depends upon the macromolecule-solvent system. The constant b depends on the molecular conformation in the solution. In normal-mode FIFFF, the retention time (tr, min) for well-retained components is approximated by [63] tr = W2 Vo/6D V
(3)
where w is the channel thickness (cm), Vc is the cross-flow rate (em 3 min-1), and V is the channel flow rate (cm 3 min'l). When D from equation (1) is substituted into equation (3), t~ becomes [47] t~ =
~
V~J2 k TV
(4)
From equations (3) and (4), tr can be directly controlled by adjusting the flow rates V, and V. Therefore, the system can be readily adjusted to suit the sample as well as to meet analysis speed and resolution goals.
BARNES and SIRIPINYANOND
196
A)
To detector flow in Crom lioN in
Porous [fit
,~loar
Mm~ane O r m flow otd
\
Porous fr#
Figure 7. FIFFF A) FIFFF schematic diagram arrangement (Reproduced from [47] with kind permission of the Dekker) B) FIFFF separation principle X represents smaller particles with higher diffusion coefficient Y is bigger particles with lower diffusion coefficient
Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry
197
Difficulties in FIFFF arise from the uneven surfaces and compressible membranes used as the FFF channel accumulation wall. This frit surfaces unevenness and membrane non-rigidity leads to some measurement uncertainty [92]. Accuracy in F1FFF, however, can be improved by using a set of calibration standards with known diffusion coefficients, hydrodynamic diameters, and by coupling the F1FFF system to an on-line detector such as a multi-angle laser light-scattering (MALLS) instrument [93-96]. The MALLS provides the absolute determination of size or molecular weight of each fraction. This FFF arrangement is referred to as symmetrical F1FFF, where the cross-flow enters the channel through the upper frit wall and leaves through the lower flit wall. Another F1FFF modification is asymmetric FIFFF, first introduced by Wahlund and Giddings [92]. In this configuration the top wall is impermeable to the liquid flow. Only one permeable wall at the bottom of the channel allows the carrier liquid to leave the channel and thus generate a crossflow. The channel and the cross flows are introduced from the inlet flow to channel. Aider its introduction in 1987, several publications using asymmetric FIFFF have since appeared [97-100]. Early channels were confined with the same rectangular geometry as the symmetrical FIFFF. The channel breadth remains constant along the entire length. With this geometry, however, a gradual fall in volumetric flow rate occurs between the inlet and outlet, became of the continuous loss through the membrane of carrier fluid as it moves downstream to the channel outlet. This leads to a gradient in the mean channel flow velocity. As a result, in the interpretation of sample component properties from the observed retention times requires correction. To overcome or compensate for this situation, a tapered channel is preferably used to maintain constant channel flow velocity. With a trapezoidal geometry where the breadth decreases toward the outlet, the velocity gradient is altered by the ratio between the breadth of the inlet and the outlet. Moreover, this breadth ratio also affects sample zone dilution [ 101 ]. Generally, the asymmetrical F1FFF experiment consists of three different steps: (1) relaxation/focusing; (2) elution; and (3) channel back-flushing. In the first step, the counteracting flows are introduced to the channel from both the inlet and outlet. The flow leaves the channel only through the membrane. By using the counteracting flows samples ar,~ focused at a certain point, called the focusing point, where the axial velocity becomes zero. The focusing point is determined by the relative rates of the forward and backward flows. The forward flow refers to the flow that is directed from the channel inlet to the channel outlet. Likewise, the backward flow refers to the flow that is introduced from the channel outlet moving upstream to the channel inlet. A
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sample is concentrated at the focusing point, where an exponential concentration profile is established during the relaxation step. Next is the elution step, when the flow enters the channel from the inlet and leaves the channel through both the membrane and the channel outlet. The former is regarded as a cross-flow and the latter is termed an outlet flow. The balance between the cross-flow and outlet flow rates is regulated by a control valve attached to the detector outlet. After the sample components have been eluted from the channel, back-flushing is required to clean the channel thoroughly. In this step, the flow enters the channel from the outlet and flushes the retained materials out of the channel [101]. Asymmetrical F1FFF provides improved separation efficiency compared to symmetrical design. Yet some limitations in versatility occur, since the cross-flow and the outlet flow rates need to be controlled precisely. Despite the complexity in mathematical and theoretical derivation, several advantages result from asymmetrical F1FFF. These include the possibility of focusing the sample into a very sharp band before separation and the simplicity of channel construction. The first feature results in higher separation resolution and improved accuracy of particle size measurements. The latter results fi'om the technical simplicity of channel construction. Only one frit (at the bottom wall) is required. Therefore, the heterogeneity resulting from the uneven permeability of the upper flit, as well as the surface irregularity, is avoided. A further advantage, pointed out by Litzen and Wahlund [97], results from decreasing the breadth along the trapezoidal channel, whereby the sample zone dilution is reduced.
2.3 Instrumentation and optimization 2.3.1 Instrumentation The experimental setup of an FFF insmanent is similar to a conventional liquid chromatograph. Instead of employing a chromatographic column as separation cell, an FFF employs a thin ribbon channel and its support. Generally, the FFF arrangement is composed of a liquid carrier reservoir, a liquid delivery system, an injection system, an FFF channel, and a detector. Typically, sample volumes are in the range of 5-200 lxL and channel volume is 1.2 mL. In this chapter only the detector is described, and complete description of other ancillary parts including the channel is found elsewhere [70,72]. Since FFF is an elution-based method, many on-line and off-line detection techniques can be used to acquire information. Most liquid chromatograph detectors can also be applied in FFF systems. Molecular absorption spectrophotometers operating in the ultraviolet or visible range are
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widely employed [69]. Fluorescence detectors also have been used, with limited numbers of applications, because most polymers and colloids do not fluoresce [69]. Therefore, samples need to be dyed or fluorophores added before the analysis. The universal refractive index detector has been applied for several macromolecules. Several light scattering detector types also have been used. These include low-angle laser light scattering (LALLS) [102], multiangle laser light scattering (MALLS) [93-96], and evaporative light scattering [103]. Direct data on molar masses of the eluted sample was obtained using LALLS in combination with a concentration detector. Nowadays, MALLS detectors have become fashionable, since they enable absolute measurement of molar masses and molecular dimensions determination. In addition, viscometric detectors can be used [ 104-106]. Recently, electrospray mass spectrometry (ESI-MS) was employed as a F1FFF detection system [107]. Fractionation of poly(styrene sulfonates) using different carrier solutions were tested. Molecular weight distribution of individual polymers was obtained by their mass fractograms. Nevertheless, salt clusters forming at high ionic strength, that leads to complicated spectra, can be troublesome. To avoid this situation, amounts of salt introduced to the electrospray should be kept below 1 mmol 11. On-line ICP-MS, pioneered by Beckett [38], has also been used. Details are given in Section 5. Many possibilities for the combination of FFF with off-line detectors also were described [108,109]. Off-line scanning electron microscopy (SEM) detection of fractionated acrylate latex from FFF channel was reported [108]. Electrothermal AAS (ETAAS) was also used for off-line detection of elemental concentrations in geological samples [ 109].
2.3.2 Optimization An FFF instrument can be operated with a constant or programmed applied field. A constant field is comparable to the isocratic elution, whereas a programmed field is analogous to the gradient elution in chromatography. For polydispersed samples, field programming during elution is necessary [110]. Generally, a high external field strength must be introduced to fractionate the least retained macromolecules or particles. Normally, well-retained species leave the channel atter an excessively long retention time. To overcome this problem, the force intensity should be decreased gradually. Typically, operation at a constant field provides the maximum resolution between the sample components at this field. Field programming can shorten the analysis time and increase the detection limit without loss of resolution, however. Once an FFF measurement has been completed, investigating the same sample separation under different experimental conditions (such as field strength) is
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worthwhile. If the same distribution is obtained at reasonably differem retemion times, the absence of artifacts is guaranteed. Lastly, to avoid samplesample interactions or overloading effects, introducing the sample with the lowest detectable concentration is desirable. Considering the F1FFF experiment, the carder liquid, membrane type, and channel and cross flowrates need to be optimized. Clearly, the membrane should have desirable molecular weight cut-off (MWCO) value. Membrane type should be carefully selected to minimize interaction between membrane surface and samples. Consequemly, the carder liquid should be well-suited for both the sample and the membrane to minimize the effect of electrostatic forces. Generally, adding salt to the carrier liquid is required to reduce the retention perturbations caused by repulsive electrostatic interactions. Since the channel and cross flow rates affect retention and fractionation power, these parameters should be optimized to obtain desirable fi'actionation resolution without excessively long retention time (or analysis time). Usually at fixed channel flow rate, increased cross-flow rate leads to longer retention time and hence improved resolution. Sample adsorption to the membrane surface may become problematic, however. Lastly, the effect of channel dimension and geometry should also be considered. This effect is beyond the scope of this chapter and described in references [97,101 ].
2.4 Quantitative Analysis by FFF Most FFF experiments exploit UV spectrophotometry as detection means, owing to its simplicity, availability, and price. For large particles (supermicron-size particles), however, dependence of the analytical response upon carder liquid composition, and the relationship between sample size and optical properties, is quite complex. For the supermicron-size range light attenuation is caused mainly by scattering (Mie scattering) rather than absorption [72]. For submicron-size particles, however, the degree of Mie scattering is negligible. Regardless ef these complications, the UV detector signal is typically used as a direct measure of the mass concentration of sample in the eluent. Nonetheless, extraction of quantitative information from an FFF experiment requires care. As a result, absolute quantification of the fractionated sample has not yet been fully demonslmted. In addition to the problems from detector calibration, complete sample recovery must be guaranteed. Practically, sample losses occur because of irreversible adsorption in different parts of the apparatus. The degree of irreversible adsorption can be controlled by changing the carder liquid composition. Reschiglian et al. have reviewed these difficulties and proposed a standardless method of analysis to determine the extinction efficiency of the particulate samples fractionated by FFF [111,112].
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The standardless analysis was defined as "a method through which a signal can be related to the concentration or quantity of the analyte by an exact equation that is reliable to allow for direct calculation of the desired quantity from a single measurement" [111]. The authors demonstrated that the lack of information on particles' optical properties were overcome [ 112]. 3. SELECTED APPLICATIONS Since a FFF separation takes place in a single phase without the participation of second phase, alteration of biological materials or other macromolecules is minimized. Owing to its open channel characteristic, shear degradation of fragile high molecular weight species becomes less significant than chromatographic techniques [47]. Molecular integrity and global structure of macromolecules are maintained. Although FFF is not well known in most fields related to biology, geology, or environmental science, sufficient applications have appeared in a few key areas to suggest its broader potential [ 113]. These applications span a billion-fold mass range from small proteins to cells and starch granules. Between these extremes, FFF has been applied to protein aggregates and conjugates, protein particles, lipoproteins, viruses, DNA, subcellular particles, milk colloids, cell lysates, polysaccharides, liposomes, bacterial cells, and pollen grains, among other materials [53]. Although quantitative analysis by FFF is possible, the majority of FFF applications have been done qualitatively. General overview of FFF in biomedical analysis was also reported [113]. In this chapter, selected applications of FFF to biological and environmental samples are reviewed. 3.1 Sedimentation FFF (SdFFF) Since its first introduction, sedimentation has been successfully applied to fractionation and characterization of particles and macromolecules in environmental and biological samples. Those applications include serum albumin microspheres [114], liposomes [115], cartilage proteoglycans [116], viruses [117,118], starch granules [119,120], DNA [121,122], bloodstream trypanosomes [123], cellular species [124], red blood cells [ 125], totoplasma gondii [126], bacteria [127-130], bacterial cell wall [131,132], and corn root membranes [133,134]. Specifically, SdFFF was exploited to analyze Creutzfeldt-Jakob disease infectious fractions [133]. In addition, protein inclusion bodies from Escherichia coli lysates were investigated [134]. Applications of SdFFF in food science and technology have also been reported. These include fat emulsion [137], pharmaceutical fat emulsion [138,139], and yeast cultivations [140]. Aggregation of nonfat dry milk proteins was also
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examined [141]. Validation procedures of SdFFF techniques for biological applications were reported [142]. For environmental samples, colloidal particles in fiver water [143,144], and aggregations of colloidal materials [145] also were investigated. Several cellular species were separated with the SdFFF with gravitational field (gravitational FFF, GFFF). These include cells [146], red blood cells [ 147-153], Pneumocytis carinii cysts [ 154], living Trichomonas vaginalis [ 155], and hemopoietic stemcell from the mouse bone marrow suspension [156]. Fractionation of starch materials from barley also was reported [ 157]. Sedimentation FFF can handle samples in both aqueous and organic solvents with satisfactory separation resolution. Polymers of larger than 106 g mol ~, and colloids or particles of larger than 30 nm can be characterized. Fractionation in the gravitational field can be applied to particles of larger than 1 ktrn. This technique has a wide range of applications including particulate materials and biological molecules. 3.2 Thermal FFF (ThFFF) Thermal FFF is applicable to samples in both aqueous and organic carder liquids. However, thermal diffusivity is strongest in organic solvents without hydrogen bonding. In other words, the separation driving force (thermal diffusion) is very weak in aqueous fluids. Therefore, ThFFF is the preferred technique for characterizing organic soluble, synthetic polymers and copolymers, as well as determination of the average molecular weight and molecular weight distributions. In addition, information about polydispersity, and the polymer Brownian diffusion coefficient also can be obtained. Since the use of organic solvents cause extensive conformational changes and even sample denaturation, the sample application range is very limited. Nonetheless, applications of ThFFF for biomacromolecules have been reported. Myers et al. first investigated fi'actionation of a water-soluble blue dextran using ThFFF in water and mixed water-DMSO carder liquids [ 158]. No retention in water was observed, owing to the weak thermal diffusion effect in water, as confirmed by Kirkland and Yau [159]. Separations of dextrans, ficools, pullulans, cellulose, and the starch polymers amylose and amylopectin using DMSO as carrier liquid were investigated [84]. These polysaccharide samples have a wide application ranges in industries and are difficult to separate by SEC. These SEC difficulties arise from sample adsorption, shear degradation and clogging of the column. Lastly, application of ThFFF to the characterization of natural rubber also was reported I160]. In stmunary, size and chemical composition can be characterized using ThFFF. The technique is favorable for lipophilic samples, but not hydrophilic
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samples. Thermal FFF is suitable for very high molecular weight macromolecules, macromolecular assemblies subject to shear degradation, and copolymers prone to surface interaction. Since the thermal diffusivity in aqueous carrier liquid is weak, applications to water soluble macromolecules are limited. 3.3 Electrical FFF (EIFFF) The E1FFF was first investigated by Caldwell et al. for its capability to separate proteins [88]. In this early investigation, albumin, lysozyme, hemoglobin, and gamma-globulin were separated. The fractionation between albumin, hemoglobin and gamma-globulin was achieved within 240 minutes. Baseline separation was not obtained with the flow rate of 60 ml min1, however. Slower carder flow rate was suggested to improve resolution. Later, human and bovine serum albumin, gamma-globulin (bovine), cytochrome C (horse heart), lysozyme (egg white), and ribonucleic acid, as well as denatta,ed proteins, were fractionated [87,161 ]. Both flexible membrane [ 161 ] and rigid membrane [87] channels were tested. Furthermore, sugars [162], colloids and particles [163] using E1FFF were separated. So far, E1FFF has been in limited use. Since it is particularly sensitive toward differences in surface charge, the technique can be applied to study the adsorption of materials to colloidal substrates [72,164]. EIFFF should be an informative tool for biological and environmental research. 3.4 Flow FFF (FIFFF) In 1977 Giddings et al. first proposed the F1FFF as a method for protein separation and characterization [91]. Proteins, plasmids, plasmid fragments, polysaccharides, unicellular algae [165], nucleic acids, viruses [166], and monoclonal antibody aggregates [167] were fractionated. Flow FFF also was applied for water soluble synthetic and biological macromolecules separation [47,168]. Linear and circular DNAs [169], and red blood cells of diverse size, shape and origin [170] were separated. Wijnhoven et al. studied the retention behavior of proteins as a function of injected mass and ionic strength using hollow-fiber FIFFF [171]. Li and Giddings evaluated a modified F1FFF technique called "membrane selective F1FFF" used for the isolation and size distribution measurement of colloids in human plasma [172]. Fractionation of lipoproteins from human serum was examined [173]. Moreover, effect of ionic strength and pH on size characterization of liposomes was investigated [174]. In addition, F1FFF was used to characterize humic acids in solution [175]. Applications of FIFFF in food and dairy technology were reported. These include characterizing high molecular weight proteins present in glutenin [176],
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wheat proteins [177,178], whey proteins, casein micelles, and fat globules in dairy products [179], and colloidal components in reconstituted skim milk [180]. Carbohydrates (like dextran in seawater [181] and pullulan [182]) also were characterized using F1FFF. Conformational change and aggregation of kcarrageenan were studied using F1FFF and MALLS [183]. Flow FFF was applied to process monitoring in biotechnology. Ribosomes and their subunits in Escherichia coli during production of glucose isomerase were monitored [ 184]. Polymeric wheat proteins were also characterized [ 185]. Asymmetrical F1FFF also has been used for proteins characterization. Wahlund and Litzen reported a rapid high performance fractionation of five proteins coveting molecular weight ranging from 12,000 to 669,000 Da with baseline separation [165]. The technique also was used for molar mass characterization of modified celluloses using on-line MALLS detection [186]. Furthermore, drug-plasma protein interaction was studied [ 187]. Like other FFF techniques, FIFFF exhibits both advantages and disadvantages. Flow FFF is universally applicable to both macromolecules and particles of biological and environmental origins. The disadvantage of the technique is due to its low resolution, which can be improved using asymmetric FIFFF configuration. 4. COMPARISON WITH SEC Size exclusion chromatography (SEC) was first established three decades ago as the standard method for polymer separation and molecular weight distribution (MWD) determination [84]. Even so, SEC is not suitable for separating ultrahigh molecular weight polymers (>1,000 kDa), owing partly to the difficulties in preparing sufficiently large porous packing to allow the permeation of large macromolecules. Shortcomings of SEC include the possibility of shear degradation of large, fragile macromolecules in porous media and column clogging by large particles. Because of the column clogging, filtration before SEC is generally required. In terms of shear degradation, random-coil macromolecules at least as large as 2 x 106 MW might be successfully separated with columns of 0.5-1am particles without serious difficulty [186]. However, the shear imposed by flow in packed columns ultimately limits the upper molecular weight separation range of fragile macromolecules. Because of these persisting problems, alternative approaches like non-chromatographic size separation techniques should be explored for certain samples (e.g., hyaluronic acid, pullulan). As mentioned earlier, FFF is a "gentle" separation technique, owing to its open-channel characteristic. Shear degradation and adsorption are minimized,
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since no stationary phase or packing material exists inside FFF channel. Global architectures of macromolecules are preserved as their native forms. Therefore, MWD information obtained from FFF is expected to be more accurate than that obtained from SEC. In addition in comparison to SEC, no MW calibration is required as long as channel dimensions are known. This is beneficial since difficulty arising from finding a good match between sample and standard macromolecules is substantial. Considering F1FFF, the cross-flow can be finely tuned permitting a single channel applicable to a very broad size range. This cannot be achieved with SEC, in which one column usually can work within only a certain size range. In other words, F1FFF has a higher upper MW limit than SEC [187]. Size exclusion chromatography gives higher resolution for low MW (< 50 kDa) materials, however. Another disadvantage of FIFFF is the band broadening. This is more serious in F1FFF than in SEC, thus the peaks resulting from an FFF separation tend to be broader than those from SEC separation. Some literature compares FFF, SEC, and electrophoresis [ 186,188]. 5. ATOMIC DETECTION
SPECTROMETRY
AS
ELEMENTAL
SPECIFIC
5.1 Literature
Field-flow l~actionation-ICP-MS (FFF-ICP-MS) is a relatively new technique for size separation with elemental analysis. Promising preliminary results have been reported for FFF coupled to ICP-MS. In 1991 Beckett first introduced the concepts and described initial experience in linking FFF separation techniques with ICP-MS [38]. According to Beckett, the initial idea of direct coupling between FFF and ICP-MS was arose during discussions with Howard Taylor of the U.S. Geological Survey in Denver, Colorado (Figure 8). Few publications applying FFF-ICP-MS have appeared since then [39-42]. In these papers natural suspended particulate matter, soil, and clay minerals were analyzed by SdFFF-ICP-MS [39-42,189,190] and by FIFFF-ICP-MS [191]. The applications of FFF-ICP-MS are summarized in Table 4.
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tN IlmtlL"ll~t
t___._ --
)k
t 9
lil m
i
Figure 8. Instrumental setup of SdFFF-ICP-MS. [Reproduced from [40] with kind permission of the American Chemical Society] A modified Sciex Elan Model 250 ICP-MS (quadrupole), Perkin Elmer was used. Table 4 Applications of FFF-ICP-MS
,,Technique Sedimentation
Samples Colloids
Sedimentation
Soil colloids
Sedimentation
Complex colloid
Sedimentation Sedimentation
Kaolin Natural colloids
Sedimentation Sedimentation
River Po particles Colloids
Sedimentation
Natural co lloMs
Flow
Colloids
Flow
Proteins
Comments Minerals and river-bornes colloids characterized AI, Fe, and Mg determined in several soil colloid samples (K monitored by ICP-AES) Kaolinite, illite and particulates characterized Off-line ETAAS tested Effect of hydrous iron oxide comings on adsorption of orthophosphate studied A1, Cd, Cr, Cu, Fe, and Pb analyzed A1, Ba, Ce, Fe, Mg, Nd, Rb, Sr, and Ti determined Adsorption studies of Cs, La, and Pb conducted A 50-mL sample introduced into the channel using opposed-flow sample concentration. Feasibility studies for biological samples
Year [Ref] 1992139] 1992 [192]
1993 [40] 1995 [ 109] 1996 [193]
1997 [41 ] 1999 [42] 1999 [190] 1999 [191]
1999 [50]
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Generally, less than 1 mg of sample is required for fractionatiorL High sample concentration may cause overloading effects in the FFF channel. With low sample concentration (e.g., < 0.5 mg mll), however, very sensitive analytical methods must be used in the subsequent chemical characterization step. Since ICP-MS provides excellent sensitivity, low detection limits, extended linear dynamic range, and also it can be applied to many elements with limited interferences, ICP-MS is an ideal elemental specific detector for FFF. In addition, ICP is a source capable of producing atoms from solid-phase particulate matter and ionizing all elements including those with high ionization potentials. The first evidence of FFF-ICP-MS application to size fractionation with elemental specific detection was published by Taylor et al. in 1992 [39]. A schematic diagram of SdFFF-ICP-MS is illustrated in Figure 8. In their work, a Babington-type pneumatic nebulizer was used to introduce and nebulize suspended particulates without the risk of clogging. In their experiment, major, minor, and trace element composition of the size-separated colloidal ( 400 kDa was also found. Several fractions were detected in a milk sample, (i.e., 3, 40, and 330 kDa). Ion fractograms of fresh strawberry juice are shown in Figure 11 as an example. For all samples studied, only monomodal or bimodal peaks were obtained. By converting the signal obtained from the ICP-MS to concentration, the concentration versus retention time curve is obtained. This curve is then integrated to give the total peak area. Subsequently, the area is translated to the amount of metal by multiplying by the flow rate and the dilution factor in the FFF-ICP-MS interface. Finally, the amount of metal elements found in the
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HAMI
HALN
a)
0.06 l
0.12 0.1 o 0.08 m 0.06
0.05
o
o
0.04
100 jam) arise from the difficulty of maintaining a homogeneous distribution of the large particles in slurry and the lower pipetting efficiency for large particles. To ensure a good repeatability of the measurements a representative number of particles must be analyzed. Slurry nebulization into atomic sources requires that both the analyte transport efficiency of the slurry particle through the sample introduction system and the atomization/excitation efficiency of that particle in the source must be identical with those of a solution. If these criteria are fulfilled then simple aqueous calibration may be used and precision of analytical results may be attained.
Slurry Sample Introduction in Atomic Spectrometry
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2.3 Slurry concentration Another important factor in the slurry technique is the slurry concentration. Slurries can be diluted, but only within a limited range as the precision may be degraded with slurries that are highly diluted. This is because of the smaller number of particles in the total volume which remain after dilution has been performed. Another factor to be taken into account is the increase in the matrix effects that can arise when the slurry concentration is increased. Conventionally, acid dissolution procedures produce solutions with a 1% sample content. Clearly, the ability to use slurries with a sample content of 20% or more yields major advantages in trace analysis. 2.4 Chemical (matrix) modification Sltm'y sampling in atomic spectrometric techniques allows the use of chemical modifiers. However, the matrix inferences are problematic owing to the different forms of the aqueous standards and biological sample suspensions. The interaction between the chemical modifier and the particles of the solid sample is closer than for direct solid sampling. Most of the work on chemical modification for the slurry technique has been carried out in order to stabilize highly volatile elements such as Cd and Pb.
2.5 Calibration techniques Different calibration methods can be employed for the direct analysis of slurries. Simple aqueous calibration may be used successfully. This technique has been used by most analysts who have achieved the desired mean particle size ( 100 ppm). A detection limit of 0.2 ppm was obtained for the determination of Co in the graphite matrix, whereas those for Co in soil and steel matrices were somewhat higher: l ppm and 20 ppm, respectively. On an absolute basis, the LODs were 30 fg for Co in graphite, 190 fg for Co in soil, and 500 fg for Co in steel. LIBS has been evaluated for depth profiling of P doping in silicon by Milan et al. [257]. The depth resolution was about 1.2 mm pulse l in this work which is sufficient for many applications. However, it should be kept in mind that its value changes even with small variations in the experimental conditions. Marquardt et al. [258] developed a fiber-optic probe LIBS system to determine the concentration of Pb in samples of dry paint. They used one optical fiber for laser excitation and a separate fiber for light collection. Both fibers were 1 ~m core diameter, 0.48 NA and the fibers were approximately 4 m long. A Nd:YAG laser operating at 1064 nm was used to produce a laserinduced plasma. Typical laser power at the probe tip for the Pb analysis was 19.0 mJ/pulse. The detection limit for Pb in dry paint were 0.014 % (w/w dry weight) and the precision of the measurements was 5 to 10%. Lucema et al. [259] presented the capability of LIBS for V determination in a xV-2TiO2-SiO3 catalyst. The microplasma was generated onto the sample surface using a pulsed Nd:YAG laser beam operating in the second harmonics (532 nm). The focusing of the laser beam on the surface was optimized to improve the signal-to-noise ratio and consequently the detection limit for the analysis. The detection limit for V was estimated to be 38 ~tg/g in 2TiO2-SiO2. The precision for measurements was better than 6% RSD in the concentration range 200-1000
g/g. Fichet et al. [260] used LIBS to investigate impurities at concentrations around 100 ppm, rapidly and in-situ at atmospheric pressure using a specially designed glove box, in two nuclear solid materials, UO2 and PuO2. The 18 elements; Ag, Al, B, Ba, Bi, Ca, Cr, Cu, Fe, Ga, In, Li, Mg, Mn, Na, Pb, Sr, and T1, have been observed in UO2 at concentrations of around 500 ppm and 12 elements; Ag, Ba, Ca, Cr, Cu, Ga, In, Li, Mn, Na, Sr, and V, in PuO2 at concentrations around 100 ppm. Detection limits of about 100 ppm were found in both matrices for the impurities studied. 6.6 Applications for advanced materials A better knowledge of the behavior of the main impurities which are usually introduced in advanced materials during production helps to reduce additional contamination. Silicon is the most extensively studied semiconductor
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mainly owing to its wide application in the microelectronics device manufacture and in the photovoltaic solar cell industry. Ciocan et al. [261] applied a laserablated microwave-induced plasma atomic emission spectrometry (LA-MIPAES) system to the direct determination of trace elemems (Mg, A1, Si, and Fe) in a high-temperature superconducting ceramic (Yba2Cu307) and the determination of a low Na concentration in high-purity natural and synthetic quartz used for the production of optical fibers. They used internal standardization for the normalization of the emission signal and calibrated with relevant spectra of standard reference samples (A1, Cu, borax glass). The measured concentrations for elements (Si, Fe, A1, Mg) by LA-MIP-AES and ICP-AES were compared to that of LIBS. Both methods revealed very similar result, e.g., for Si, 2670 + 330 ~tg/g by LA-MIP-AES and 3300 + 300 ~tg/g by LIBS. The concentrations for other elements in YbaECU307 were 114 + 40 ~tg/g for Fe, 114 + 8800 ~tg/g for A1, and 26 + 5 ktg/g for Mg. For A1 a strong inhomogeneity was found in differem spots of the target. In this study, internal standards such as a known concentration of Cu were used for the determination. The concentration of Na on natural and synthetic quartz was 0.28 to 2.0 ~tg/g. The detection limit for Na was estimated as 40 ng/g by adopting the square root of 3-6. The precision of the measurement was not as good as ICP-AES, but was comparable to that of LA-MIP-AES. Ottesen [262] applied the LIBS technique to analyze the elememal composition of contaminants found on electronic microcircuits fabricated on alumina substrates and obtained spatially resolved data with some degree of depth profiling information. One of the potential uses of LIBS in the field involves depth profiling of surface coatings. The composition of each individual layer, particularly at the interface, was much more informative than the composition of the average over a range of depths. Hidalgo et al. [263] investigated the emission spectra of a laser-generated plasma from titanium dioxide anti-reflection coatings in solar cells. A method for measuring time TiO2 films between 40 and 400 nm within the typical values used in solar cells, based on the LIBS technique has been developed. A pulsed nitrogen laser at 337.1 nm was used with a pulse width of 10 ns and a laser fluence of 8.6 J/cm 2 on the sample. The capability of LIBS to resolve complex depth profiles was also demonstrated by Vadillo and Lasema [264] using electrolytically deposited brass samples. Ablation depths of 6.5 ng per pulse were obtained, which imply absolute detection limits of the order of fg per pulse for an element present in the sample at a concentration of a few ppm. Vadillo et al. [265] studied the lateral and depth resolution for surface analysis using LIBS. A pulsed nitrogen laser at 337.1 nm (3.65 J/cm2) was used to irradiate solar cells employed for photovoltaic energy production. The emission lines for Ag(I) at 545.9 mm,
Laser-Induced Breakdown Spectrometry
347
C(II) at 588.9 nm and Si(II) at 634.7 nm were detected clearly by a single-shot LIBS spectrum of a solar cell. Further research by Romero and Laserna [266] was performed to generate selective chemical images for Ag, Ti, and C from silicon photovoltaic cells with a multichannel LIBS system. Both surface and depth distributions were amenable with this approach. Lateral resolution of 80 mm and depth resolution of better than 13 nm for TiO2 coatings were achieved in this work.
6.7 Miscellaneous applications Experimental studies on laser-ablated ZrC were performed by Wantuck et al. [267] for the investigation of fuel corrosion diagnosis in nuclear fuel. Monitoring of the fuel corrosion products is important not only for understanding corrosion characteristics, but to assess the performance of an actual, operating nuclear propulsion system. Anglos et al. [268] employed the LIBS technique for the in-situ analysis of pigments used in painting. Appropriate emission lines for the identification of the metallic elements in the pigments examined were proposed. Furthermore, a test of an 18th century oil painting was examined by LIBS and the different pigments used in the original and in the restored part of the work were clearly identified. Anglos [269] reviewed the use and potential of LIBS in art and archaeology. The LIBS technique has also been applied as a real-time diagnostic technique for the laser cleaning of natural marble surface by Maravelaki et al. [270]. They demonstrated that LIBS can be used for the on-line control to the extent of laser cleaning at each spot of the surface by analyzing the spectra of the plasma emission. The LIBS technique has been successfully applied as an on-line diagnostic technique in the laser cleaning process of polluted limestone by Gobemado-Mitre et al. [271]. Monitoring of the relative intensities of selected emission lines such as Ti, Fe, and Si, can be used as an indicator of the extent of the cleaning process. Therefore, an efficient on-line control of the laser cleaning process of limestone has been achieved by monitoring the consecutive LIBS spectra while the encrustations are being removed. Kim et al. [272] applied LIBS for compositional mapping of a commercial printed circuit board. A Nd:YAG laser beam with second harmonic module was focused on the solid surface with the optimum energy of 5 mJ. The authors successfully accomplished their study for a few millimeters size of samples. This is not the sample size for the scanning electron microscope (SEM)/energydispersive X-ray (EDX) technique. Sattmann et al. [273] studied the LIP emission of the visible and near-UV spectral region of polyethylene (PE), polypropylene (PP), polyethylene (PET) and polyvinyl chloride (PVC) polymer samples to evaluate the feasibility of LIBS for the identification of different
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polymer materials. Spectral features were measured with the use of the 725.7 nm chlorine wavelength, the 486.13 nm lib line, and the 247.86 nm carbon line. The evaluation with neural networks of the spectral data measured with clean samples enables accurate identification in the range of about 93 to 96% for PE and PP and of >99% for PET and PVC. The application of LIBS for certain cases, such as a high-speed separation of washed PET and PVC bottle. 7. CONCLUSION This chapter has shown the potential and versatility of L1BS as an analytical technique to determine elements or metals in a wide variety of samples, including solids, liquids (solutions) and gases. It has not achieved widespread use in the biological and clinical area, in part, due to the lack of commercial instrumentation, its somewhat reduced sensitivity compared to conventional and competitive analytical techniques such as those described in the other chapters of this volume. It lack of ability to determine the chemical form or species is also a drawback in the biological and clinical area. The greatest and most cutting edge determination in biological or clinical samples is in this area. However, LIBS continues to attract the interest of scientists and researchers throughout the world. It will become more widespread in the cbiolical and clinical area in the near future. It is not uncommon to find sessions devoted to this technique at scintific meetings and more recently a complete conference in this area. A special issue of Applied Optics devoted to LIBS is currently underway and should be published late in 2002/early 2003. LIBS continues to thrive. REFERENCES 0
2. 0
*
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246. M. Essien, L.J. Radziemski, L.J. and J. Sneddon, J. Anal. Atom. Spectrom., 3 (1998) 985. 247. E.D. Lancaster, K.L. McNesby, R.G. Daniel and A.W. Miziolek, Appl. Optics, 38 (1999) 1476. 248. D.W. Hahn, Appl. Phys. Lea., 72 (1988) 2960. 249. M. Tran, B.W. Smith, D.W. Hahn and J.D. Winefordner, Appl. Spectrosc., 55(11) (2001) 1455. 250. J. Uebbing, J. Brust, W. Sdorra, F. Leis and K. Niemax. Appl. Spectrosc., 45 (1999) 1419. 251. D. Franzke, H. Klos and A. Wokaun, Appl. Spectrosc., 46 (1992) 587. 252. E. Stoffels, P.V.D. Weijer and J.V.D. Mullen, Spectrochim. Acta, 46B (1991) 1459. 253. L.C. Jensen, S.C., Langford, J.T. Dickinson and R.S. Addleman, Spectrochim. Acta, 50B (1995) 1501. 254. J. Blacic, D. Pettit, D.A. Cremers, and N. Roessler, N. In Lunar and Planatary Inst., Workshop on advanced technologies for planatary instruments, (1993) Part 1 and 2. 255. Y.Y. Yoon, T.S. Kim, K.S. Chung, KY. Lee and G.H. Lee, Analyst, 122 (1997) 1223. 256. I.B. Gomushkin, J.E. Kim, B.W. Smith, S.A. Baker and J.D. Winefordner, Appl. Spectrosc., 51 (1997) 105. 257. M. Milan, P. Lucena, L.M. Cabalin and J.J. Lasema, Appl. Spectrosc.,52 (1998) 444. 258. B.J. Marquardt, B.M. Cullum, J.J. Shaw and S.M. Angel, Proc. SPIE-Int. Soc. Opt. Eng., 3105 (1997) 203. 259. P. Lucema, L.M. Cabalin, E. Pardo, F. Martin, L.J. Alemany and J.J. Lasema, Talanta, 47 (1998) 143. 260. P. Fichet, P. Mauchien and C. Moulin, Appl. Spectrosc., 53 (1999) 1111. 261. A. Ciocan, L. Hiddemann, J. Uebbing and K. Niemax, J. Anal. Atom. Spectrom., 8 (1993) 273. 262. D.K. Ottesen, Appl. Spectrosc., 46 (1992) 593. 263. M. Hidalgo, F. Martin and J.J. Laserna, Anal. Chem., 68 (1996) 1095. 264. J.M. Vadillo and J.J. Lasema, Talanta, 43 (1996) 1149. 265. J.M. Vadillo, S. Palanco, M.D. Romero and J.J. Lasema, Fresenius J. Anal. Chem., 355 (1996) 909. 266. M.D. Romero and J.J. Lasema, Anal. Chem., 69 (1997) 2871. 267. P.J. Wantuck, D.P. Butt and A.D. Sappy, Los Alamos National Laboratory Report No. LA-UR, 92 (1992) 1557. 268. D. Anglos, S. Couris and C. Fotakis, Appl. Spectrosc.,51 (1997) 1025.
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269. D. Anglos, Appl. Spectrosc., 55(6)(2001 186A. 270. P.V. Maravelaki, V. Zafiropulos, V. Kilikoglou, M. Kalaitzaki and C. Fotakis, Spectrochim. Acta, 52B (1997)41. 271. I. Gobemado-Mitre, A.C. Prieto, V. Zafiropulos, Y. Spetsidou and C. Fotakis, Appl. Spectrosc., 1998, 51, 1125e. 272. T. Kim, C.T. Lin and Y. Yoon, J. Phys. Chem. B, 102 (1998) 4284. 273. R. Sattmann, I. Monch, H. Krause, R. Noll, S. Couris, A. Hatziapostolou, A. Mavromanolakis, C. Fotakisi, E. Larrauri and R. Miguel, Appl. Spectrosc.,52 (1998) 456.
Chapter 7
Application of graphite furnace atomic absorption spectrometry in biological and clinical samples Joseph Sneddon I and David J. Butcher 2 1-Department of Chemistry, McNeese State University, Lake Charles, Louisiana 70609, USA and 2-Department of Chemistry and Physics, Western Carolina University, Cullowhee, North Carolina 28723, USA I. INTRODUCTION Atomic absorption involves the measurement of the reduction of intensity of optical electromagnetic radiation, from a light source, following its passage through a cell containing gaseous atoms (the atom cell). Atomic absorption spectroscopy generally refers to the study of fundamental principles of this phenomenon, whereas atomic absorption spectrometry (AAS) refers to its use for the quantitative determination of metals in a wide variety of samples, although these terms are often used interchangeably. AAS is applicable for the determination of most metals (almost all metals and metalloids and some non-metals, approximately 70 elements in the Periodic Table), in a wide variety of samples including biological, clinical, environmental, food, and geological, and hence is one of the most commonly used analytical techniques for elemental or metal determination. Two types of atom cells have been commonly used for AAS. The flame is widely used became of its ease of use for elemental analysis at the parts per million (ppm) (ktg/mL) level. However, the use of a graphite furnace as the atomizer is used when a limited sample volume is available or lower analyte concentrations (parts per billion) (ppb) (ng/mL) level are present. In this case, the technique is commonly referred to as graphite furnace atomic absorption spectrometry (GFAAS) or electrothermal atomization atomic absorption spectrometry (ETAAS). An earlier name of carbon furnace atomic absorption spectrometry (CFASS) is not widely used. The purpose of this chapter is to provide a brief and general overview of the technique including the basic principles and theory, and insmunentation. This will show the reader the potential of GFAAS in the determination of metals in clinical and biological samples. Selected and recent applications are highlighted.
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1.2 S P E C T R O S C O P Y Spectroscopy is defined as the interaction of electromagnetic radiation (light) with matter. Electromagnetic radiation is described as having both wave and particle properties. Wave properties include frequency (v, Hz), wavelength (~,, meters), velocity, and amplitude. Light is also considered to be composed of particles called photons and have a characteristic energy (E, Joules). The relationships between energy, frequency, and wavelength are given by: E = hv = h--c-c
(1)
2 where c is the speed of light in a vacuum (2.99792 x 108 m/s) and h is Planck's constant (6.62608 x 10.34 J s). The electromagnetic spectrum covers a wavelength range of over 14 orders of magnitude, including the gamma ray, X-ray, ultraviolet, visible, infrared, microwave, and radio frequency regions. For atomic absorption spectrometry, we will focus on a relatively limited region of the spectrum between 180 and 900 nm (ultraviolet, visible, and near infrared). These wavelengths are involved in electronic transitions of valence electrons. 1.2.1. I n t r o d u c t i o n to a t o m i c s p e c t r o s c o p y
It is beyond the scope of this chapter to provide a detailed description of the process of atomic spectroscopy. These details are available elsewhere [1, 2, 3] but a short review is given for completeness. Atomic spectroscopy involves the interaction of light with gaseous atoms. A device converts a sample (usually a solution) into gaseous atoms and is called an atom cell. Typical atom cells include flames, plasmas, and graphite furnaces. There are three basic types of atomic spectroscopy: atomic emission, atomic absorption, and atomic fluorescence. While the three processes are related they do offer three unique analytical techniques. In order to introduce these phenomena, we will initially consider an atom with only two electronic energy states, in which the ground (lowest energy) state is designated 0 and the excited state as 1. It can generally be assumed that under normal conditions the majority of atoms are in the ground state. Atomic emission (AE) involves the transfer of energy, usually as heat, from the atom cell to the atom to promote a valence electron in the atom from the ground state to the excited state. The atom then may emit a photon, and deactivate to the ground state (emission). The energy of the photon is equal to the difference in energy between the states. This process is called an electronic transition.
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Atomic absorption (AA) involves the transfer of the energy of a photon to an atom (absorption) to promote a valence electron in the atom from the ground to the excited state. In order for absorption to occur, the energy of the photon must be identical to the difference in energy between the lower and higher energy levels of the atom. Atomic fluorescence (AF) involves the excitation of atoms from a lower energy state (usually the ground state) to a higher energy state by light, followed by the emission (fluorescence) of a photon to deactivate the atoms. AFS can be considered to be a combination of atomic absorption and emission because it involves radiative excitation and de-excitation. Atomic spectra are characterized by their relative simplicity, typically consisting of narrow lines, which correspond to the limited number of possible energy levels. Each element has a unique set of energy levels and hence a unique spectnnn. At the temperatures of the atom cells used for atomic absorption (15003000~ the vast majority of atoms are present in the lowest energy level, called the ground state, and consequently the most sensitive lines involve transitions from the ground state, which are called resonance transitions. A transition from the lowest energy state is called a first resonance transition. The full width at half maximum (half-width) of most atomic lines is typically 0.01 - 0.05 nm, which is much narrower than liquid-phase molecular bands, whose widths are typically 10100 nm. The absence of vibrational or rotational levels in atoms results in the narrow widths of atomic lines. 1.3 GFAAS A N A L Y T I C A L SIGNAL: ABSORBANCE The fundamentals of a quantitative atomic absorption measurement are illustrated in Figure 1. In the ideal case, a monochromatic light beam from the source of intensity (Io) enters a cell containing the gaseous analyte. The transmitted beam (I) then passes into a detection system and converts the light beam into an electrical signal. If analyte is present in the cell, then the transmitted beam is less intense than the incident beam. On the other hand, if no analyte is present in the cell, then the incident and transmitted beams are equal. The ratio of the transmitted beam to the incident beam is defined as the transmittance, T (unitless):
T= •
Io
(2)
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Figure 1. Schematic diagram of atomic absorption process, where b is the pathlength of the atom cell, Io is the incident intensity, and I is the transition intensity which represents the fraction of light transmitted through the cell. Alternatively, the percent transmittance, % T, is defined as % T = I x 100% Io
(3)
Transmittance values range from 0, in which case no light passes through the cell, meaning a very high concentration of analyte atoms are present in the cell, to 1, in which no atoms are present in the cell. There is not a linear relationship between transmittance and concentration, and hence quantitative measurements are usually made using absorbance, A (unitless): A
=
-logT
-
I=log -lOglo
~
(4)
Notice that the absorbance increases as transmittance decreases, indicating as more atoms are present in the cell, the absorbance increases. Absorbance is a unitless number, typically, 0 to 2, with optimum precision between 0.1 and 0.5. The quantitative relationship between absorbance and concentration (c, g/L solution) is described by the Beer-Lambert Law:
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A : abc
(5)
where a is a constant called the absorptivity (L g-I cm-l) and b is the pathlength of the cell (cm). The magnitude and units of absorptivity are determined by the units for the pathlength and concentration. The absorptivity and concentration are related to the absorption coefficient k (cm l ) by a : 0.434 k
(6)
c
Atom cells for AAS typically employ a relatively long illuminated volume because of the direct proportionality between absorbance and pathlength. The GFAAS absorption signal differs from that of flame AAS because of the temporal variation of the atom population, producing a transient signal. A discrete amount of sample (10-50 laL) is introduced into the graphite furnace, which is heated to a series of temperatures for specified times. This process is called an atomization cycle. During the atomization step, the tube is heated to a sufficiently high temperature to convert the sample into gaseous atoms. A plot of absorbance versus time shows no signal at relatively low temperatures, before the atoms have been produced, an increase in absorbance as atoms are formed, and a decrease in absorbance as atoms are swept out of the atom cell. 1.4 THE NATURE OF THE TRANSIENT GFAAS SIGNAL: M E C H A N I S M OF ATOM F O R M A T I O N IN A GRAPHITE FURNACE
Within a few years after the development of GFAAS as an analytical technique in the late 1950s and 1960s, questions were raised regarding the chemical and physical processes involved in the conversion of the (commonly) aqueous sample into gaseous atoms, followed by their removal from the furnace. Although a considerable amount of information has been obtained regarding these processes, a commercial graphite furnace is a sufficiently complex system that uncertainty exists regarding the mechanism of atom formation for many elements. A very general mechanism for atom formation is Metal Salt (aq)
('), Metal Salt (s)
Metal (s).-(4)~ Metal (g)
(2), Metal Oxide (s)
(3),
(7)
A graphite tube is typically 20-30 mm in length and 3-6 mm in diameter. The tube is surrounded by argon to prevent combustion in air at elevated
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temperatures. The sample is introduced into the furnace through a dosing hole (1-3 mm in diameter). In many cases, chemical compounds called chemical modifiers are also added to improve the sensitivity or accuracy for a given analyte. The temperature of the tube can be controlled from ambient up to approximately 2700~ with heating rates up 1500~ It has been shown to be beneficial for many elements to insert a platform into the tube onto which the sample is placed. In our general mechanism (Equation 7), we have chosen to show vaporization of the metal as a starting point for the mechanisms, although some metals may vaporize as a compound. The first step involves the relatively straightforward removal of solvent (usually water) from the sample. The remaining steps include chemical/physical surface processes, such as homogeneous or heterogeneous solid-solid interactions, solid-phase nucleation, and diffusion into graphite; heterogeneous gas-solid interactions, i.e., adsorption/desorption and reaction of molecules with the wall to form atoms; homogeneous gas phase reactions; and processes by which analyte leaves the furnace. Steps (2) and (3) involve the chemical and physical processes which occur on the surface of the graphite tube. These processes are probably the least well understood because of the unavailability of a technique to monitor species on the surface of a commercial tube during a heating cycle. In fact, many of the techniques employ graphite substrates different from those used in GFAAS, or involve measurements made at room temperature. In spite of their limitations, these methods have revealed significant information regarding the mechanism of atom formation. 1.5 INSTRUMENTATION A block diagram for a graphite furnace atomic absorption instrument is given in Figure 2. The graphite tin,ace serves as the atom cell, which converts liquid or solid samples into gaseous atoms. A power supply provides current to control the temperature of a graphite tube between ambient and approximately 3000~ A light source is used to radiatively excite analyte atoms in the tube. The quantity of light absorbed is recorded by a detection system to do quantitative analysis. The detection system is composed of a wavelength selector, which is used to separate the analytical wavelength from other wavelengths emitted by the source, and a detector, which converts electromagnetic radiation into an electric signal. A signal processor amplifies the signal and sends it to a readout device, which in modem instrumentation is a microcomputer. In addition, the microcomputer serves to control other components of the instrument. A background correction system is required to correct for attenuation of the source by molecular absorption or scatter and perform accurate quantitative analysis. A discussion and description of the complete instrumentation for GFAAS is beyond the scope of this chapter and is
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Graphite Furnace Atomic Absorption Spectrometry
described elsewhere [1, 2, 3]. This chapter will concentrate on the graphite furnace or electrothermal atomizer part of GFAAS instrumentation.
i" _ '1 ~engtht " ..=[~etector ~ [Signal Gra Light acete~.' I "I~Ve~i lPr~176 [[Readout ~1 Device ! Fun~hi Furnace Power
Supply
Figure 2. Block digram of the instrumentation for graphite furnace AAS. reference [ 1] for details
See
1.5.1 Graphite Furnace Since the initial use of a graphite fiamace with atomic absorption spectrometry by L'vov in 1959, considerable development in graphite furnace materials and design, called modem fitmace technology, has occurred that has increased the sensitivity and accuracy of the atomizer for practical analysis. This section focuses upon a description of successful furnace design. The graphite furnace serves as the atom cell, whose function is to convert the analyte in a sample (solid, liquid, or gas) into gaseous atoms that can be monitored spectroscopically. A schematic diagram of this device, which is also called an electrothermal atomizer (ETA), is given in Figure 3. A graphite tube, typically 3-6 mm in inner diameter and 20-40 mm in length, is heated resistively by a high current (up to several hundred amps), low voltage (6-12 V) ac power supply. Sample introduction into the tube is performed through the dosing hole in the center of the fiamace length. The source beam passes through the tube in the axial direction and on to the detection system. The temperature of the tube can be controlled from ambient to 2700~ with a precision of + 10~ up to 200~ and ~:
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J. SNEDDON and D.J. BUTCHER
50 ~ at the higher end of the range. The tube is mounted in graphite inserts and are held in place by brass or stainless steel water-cooled electrodes that are electrically connected to the power supply. The internal and external walls of the tube are bathed in a purge gas, usually argon, to exclude oxygen and prevent combustion of the graphite surfaces. Argon is preferable to nitrogen because the latter forms compounds with several elements (e.g., aluminum) and toxic CN gas. Removable quartz windows serve to prevent intrusion of oxygen into the fiu'nace through the optical path. The internal flow of gas is usually turned off ("gas-stop") when the analytical measurement is performed to maximize the time that the analyte atoms are present in the tube ("residence time") and the sensitivity. The internal gas flow generally may be specified by the analyst during each step of the furnace heating cycle. In addition, it is usually possible to switch to an alternative gas (e.g., air or oxygen for oxygen ashing) at specified times. The temperature of the tube is controlled two ways. During the majority of the fm'nace cycle, the tube temperature is regulated by the amount of voltage (current) applied. However, in order to obtain the best sensitivity and accuracy, the ~ce is usually heated with the maximum possible heating rate (> 1000~ to a temperature just above the minimum required to completely atomize the analyte. An optical temperature sensor is used to monitor the tube wall temperature in most modem commercial designs during maximum power heating. When the tube temperature exceeds the selected atomization temperature, the optical pyrometer ttmas off the maximum power heating and temperature control is retraced to the fia'nace power supply. Alternatively, some instruments use the optical temperature sensor to control the temperature of the tube during the pyrolysis and atomization steps.
1.5.2 Graphite tube material and design Early GFAAS work in the 1970s employed relatively large, longitudinallyheated tubes (50 mm in length, 8 mm in diameter) which had the advantages of being able to accommodate large sample volumes and relatively easy alignment of the source through the tube. Longitudinally heated furnaces are commonly referred to as Massmann furnaces, who first described this design in the late 1960"s. However, large fia'naces cannot be heated rapidly and tend to heat unevenly, which allows condensation of material in the cool areas. During the early period, open graphite atomizers were also employed for AAS. These atom cells provide very high sensitivity for simple aqueous solutions, but are unsuitable for analysis of complex sample matrices. Atomization occurs into a relatively cool environment that induces molecule formation between the analyte and its matrix. The most commonly used material for early graphite tubes was polycrystalline graphite (electrographite), which is porous, allowing diffusion of
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analyte molecules into the material, and is relatively reactive towards metals, which may cause interferences. The porosity and reactivity of electrographite may induce tailing, memory effects, or incomplete atomization. This material is particularly unsuitable for the determination of several elements (e.g., vanadium, titanium, molybdenum) that form stable, involatile carbides.
i
GraphiteFumace I
;I
DO~eng l, power supp,, Water-cooled
\ I
Io0t,ca, I I I
I Temp.
Ix ISensor ]
Electrode\
I
Quartz W~nd~
Source ......... ...~ .."~.1..................... [~....'~.. DeTOction / System raphite~ '
-
--I
,be
I
~ Cooling Internal ~ Cooling External Water Gas Water Gas Exit Flow Inlet Flow
(i~ Argon Supply
Figure 3. Schematic diagram of graphite furnace atomizer Modem graphite furnace systems employ graphite tubes (20-30 mm in length; 3-6 mm in diameter) that can be rapidly heated (> 1000~ to reduce interferences. A high heating rate also allows vaporization with a lower final atomization temperature, which is particularly advantageous for involatile elements. The problems with electrographite can be greatly reduced by the formation of a layer of pyrolytic graphite (50 lam) on the surface of a polycrystalline tube. Pyrolytically coated graphite tubes are produced by heating an electrographite tube in 5 % methane/argon. The dense layer of pyrolytic graphite serves to reduce diffusion of analyte into the graphite and chemical
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J. SNEDDON and D.J. BUTCHER
reactivity. Carbide-forming elemems should only be determined with pyrolytically coated tubes to obtain acceptable sensitivity and accuracy. A variety of other materials have been employed as tube substrates for graphite finnace AAS which include total pyrolytic carbon, glassy carbon, metal furnaces (e.g., tantalum, tungsten), metal liners (thin metal sheets), and metal impregnated into graphite. In general, these approaches have not been proven to superior to pyrolytically coated tubes for a wide variety of applications, and hence they have been employed in a relatively small number of laboratories. Up until recently, all commercial graphite furnaces were heated by a longitudinal flow of current through the furnace. Recently it has been shown that the temperature of the gas phase in a longitudinally heated graphite furnace may vary as much as 1200~ fi'om the center to the ends. The relatively cool temperatures at the ends of the furnace, caused by contact with the water cooled electrodes, may allow analyte atoms to condense, or analyte-containing molecules to form, in the colder areas. Condensation has been shown to induce chemical interferences. One approach to minimize these effects is the use of a transversely-heated graphite atomizer (THGA) with integrated contacts. The temperature gradients are reduced with these atomizers compared to longitudinally heated fiamaces, resulting in reduced interferences compared to the Massmann design. However, it has been postulated that the analyte and matrix may condense in cool regions in a THGA. In addition, commercially available THGAs (Perkin-Elmer Corporation, Norwalk, CT) are employed in a longitudinal magnetic field for ac Zeeman effect background correction that has dictated the use of a relatively short tube length (18 mm) that resulted in a reduction of the peak area sensitivity by a factor of two. In order to reduce interferences and improve the sensitivity, pyrolytically coated carbon disks with small (3.2 mm) apertures, called end caps, have been inserted in the end of the tube. The use of end caps reduces the rate of diffusion of the analyte out of the tube, and provides comparable peak area sensitivity to longitudinally heated fin'naces. In addition, they increase the temperature of the gas phase at the ends of the tube, reducing condensation and the potential for interferences.
1.5.3 Furnace Heating Cycle In order to make an analytical measurement by GFAAS, it is necessary to set up a program to control the temperature of the fiu'nace following introduction of the sample. An optimized temperature program allows vaporization of non-analyte (matrix) species, such as solvent and organic matter, before atomization of the analyte, in order to reduce potential interferences. It is also essential that all analyte
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vaporize in a temporally reproducible way, and that all is removed by the end of the fumace program. A typical fiwnace program consists of the following steps: (1) sample introduction; (2) a dry step, to remove solvent (usually water) from the sample; (3) a pyrolysis step (also called char or ash) to remove organic and other volatile materials in the sample before the analyte is vaporized; (4) a cool-down step to allow the fitmace to reach ambient temperature; (5) an atomization step in which the analyte is atomized and the integrated absorbance recorded; and (6) a clean step to remove any residual material from the graphite tube. With modem instrumentation, sample introduction is normally done with an autosampler. For the introduction of liquid samples, which are used in the vast majority of GFAAS work, the autosampler consists of a mechanical arm attached to a piece of plastic tubing and a series of standard and sample solutions in cups positioned in a sample tray. One end of the tubing is connected to a pump and a deionized water supply, while the other end is connected to a mechanical arm that may be inserted into the solutions. The sample tray rotates, and moves laterally, to allow sampling of each of the solutions in the tray. The tubing is inserted into a sample, and the pump draws a user-specified volume into the tubing, and the mechanical arm moves the tubing through the dosing hole into the graphite tube to deliver the sample. Generally sample volumes of 5-50 ~tL are employed; most modem programs use 10-25 laL. Most autosamplers allow introduction of up to three solutions into the furnace (e.g., sample, standard, chemical modifier). The autosampler then rinses the tubing with deionized water to clean it, and the heating cycle of the furnace begins. It is usually necessary to observe the sample introduction process the first few samples of the day to ensure the solution is precisely and accurately delivered into the furnace. The end of the tubing normally should pass through the center of the dosing hole and be positioned 2-3 mm above the graphite substrate during sample introduction. Normally the position is set manually by the user, before a graphite furnace cycle is initiated. A dentist's mirror, or a solid state charge coupled device (CCD) camera available on some instruments, is used to observe the sample introduction process. The CCD camera has been used to optimize set-up and alignment of the graphite tube and to ascertain that all of the liquid has been deposited correctly in the tube. Phase transitions of the injected samples may be observed during the dry and pyrolysis steps. A second type of autosampler employs deposition of the sample as an aerosol spray. This autosampler has the advantage that it can also be used for flame AAS, and is well-suited for use with a graphite tube that has been preheated, which decreases the time required for the dry step and hence the analysis time. However, aerosol deposition requires several milliliters of sample (although only 20-50 ~tL
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are introduced into the furnace), and is problematic with samples that are viscous or contain undissolved material. The use of an autosampler has three principal advantages for GFAAS. Precision is usually degraded with manual pipetting because it is difficult to reproducibly dispense the sample in the same location. Manual pipetting may cause introduction of material on the edge of the dosing hole of the tube. Typical precision values of several percent are obtained with manual sample introduction, as compared to one percent or less with an autosampler. Second, each heating cycle requires 2-3 minutes, and hence manual operation is unproductive for the operator. Third, pipette tips employed in manual pipetting may introduce contamination. The dry step serves to remove liquid present in the sample. With aqueous samples, the furnace is heated to 110-150~ lower temperatures are used with wall operation and higher temperatures with a probe or platform. The tin,ace is heated relatively slowly (2 - 20~ to a temperature just high enough to completely remove the solvent without spattering. Improper drying can lead to poor precision. It is normally prudent to observe the drying step for the first few furnace cycles of the day, using a mirror or camera system described above. The pyrolysis step serves to remove non-analyte (matrix) components of the sample. A moderate heating rate of 50-200~ is generally used during this process. It is usually desirable to use as high a pyrolysis temperature as possible without vaporization of the analyte, and determination of the optimum temperature is generally required for each element and type of sample. Pyrolysis temperature optimization involves measuring the absorbance from a standard or sample at a fixed atomization temperature with a variety of pyrolysis temperatures. Usually it is necessary to do temperature optimization for standards and samples to ensure the pyrolysis temperature is optimum for both. It is usually desirable to use a pyrolysis temperature of at least 1000~ to minimize interferences. The use of chemical modifiers allows the use of relatively high pyrolysis temperatta'es, even for volatile elements (e.g., lead). The introduction of air or oxygen into the fimmce during this step allows ashing in the graphite tube (oxygen ashing). Oxygen ashing allows more complete vaporization of some organic matrices, such as blood, and prevent formation of carbonaceous residue which cause interferences. Oxygen ashing is performed at temperatures below 800~ to prevent combustion of the furnace. Following the pyrolysis step, the furnace temperature is returned to ambient using a cool down step. The use of a cool-down step before atomization helps ensure atomization of the sample into a hot environment, which has been shown to reduce interferences.
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In the atomization step, the temperature of the graphite tube is rapidly increased with maximum power heating (> 1000~ to just above the temperature required to atomize the analyte (1600-2700~ The analytical measurement is performed during the atomization step, a transiem absorption signal is obtained. Optimization of the atomization temperature is generally required for standard solutions and samples, and involves the measurement of absorbance of a standard or sample with a fixed pyrolysis temperature and a series of atomization temperatures. The clean step serves to remove residual material from the sample in the fin'nace. The tube is usually heated at a few hundred degrees per second up to 2500-3000~ After the clean step, the tube is allowed to cool to ambient temperature, and the cycle is initiated again. Typical fim~ce programs require 2-3 minutes per analysis. Fast fimmce programs have been developed in order to approximately double the sample throughput of GFAAS. Typically the sample is introduced into a hot (150-200~ furnace, and short (5 s) pyrolysis steps are used at relatively high temperatures (> 1000~ Good results have been obtained for a number of elements in relatively easy matrices. More work needs to be performed to determine whether fast programs can be widely used for routine analysis. 1.5.4 Methods of Atomization
Early commercial atomic absorption atomizers heated at relatively slow rates (500-800~ with sample introduced on the wall of the tube, and hence atomization occurred as the fiwnace was heating to its final temperature. Under these conditions, atomization occurred into a tube whose temperature varied fi'om fiwnace cycle to furnace cycle, and was not isothermal along the length of the tube, with the center several hundred degrees hotter than the ends. Wall atomization was shown to be less suitable for real sample analysis with volatile elemems because these temperature variations were shown to degrade precision and induce the formation of analyte-containing molecules that cause chemical interferences, although wall atomization is preferable for extremely involatile elements. Several approaches have been investigated to ensure atomization occurs into a relatively high temperature environment. The most commonly used approach is the L'vov platform or simpley called platfoma, in which a sample is introduced onto a small graphite shelf (usually pyrolytically coated polycrystalline graphite or totally pyrolytic graphite) inside the tube. Currem does not pass directly through the platform, and hence it is primarily heated radiatively by the tube walls. Consequently, the use of a platform with a rapidly heated furnace ensures that atomization occurs after the tube, and the gas inside it, have reached a relatively constant temperature after maximum power
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heating. This means that atomization occurs into a hot environment, minimizing interferences. Today most manufacturers offer graphite tubes with integrated platforms. Transversely heated graphite tubes include a pyrolytically coated platform machined from the same piece of graphite. Contact between the tube and platform is minimized to reduce heat transfer through direct contact. A second approach is the delayed atomization cuvette (DAC), in which a graphite tube is modified so that the outer diameter at the middle is greater than at the ends, with a constant inner diameter. In a delayed atomization cuvette, sample is introduced into the middle, thicker region. The thinner ends of the fin'nace are heated more rapidly than the center, allowing vaporization into a relatively hot environment. In general this approach does not seem to be as effective at reducing interferences as the L'vov platform. Probe atomization involves the use of a graphite probe that is inserted into and removed from the tube by a stepper motor. Sample is deposited onto the probe with the probe inside the fiamace, and the sample is dried and ashed. The probe is then withdrawn from the finaaace which is subsequently heated to the atomization temperature. The probe is rapidly reinserted into the furnace, allowing atomization into a hot environment. Probe atomization has not been as widely employed as platform atomization, probably because of the added complexity of the instrumentation, and because the insertion of the cool probe into a hot tube cools the vapor, which prevents isothermal vaporization. In addition, the probe hole provides an additional avenue for loss of analyte. A two-step fiimace employs two power supplies, one to heat the graphite tube transversely, and the other to heat a graphite cup, just below an aperttwe in the tube, into which sample is introduced. The tube is heated to the atomization temperature, and subsequently the cup is heated to vaporize the analyte into the isothermal tube. Imerestingly, this design is very similar to the first graphite fumace instrument described by L'vov. The design has not been available in commercial instrumentation, probably because of the additional cost of two power supplies, and has relatively small advantages for most analytical applications compared to conventional atomization with a transversely heated furnace, although two-step fia'naces have been employed for ftmdamental studies.
1.6 Sample Preparation and Sample Introduction Sample preparation involves the conversion of a sample into a form that is suitable for analysis, which in general, is into a solution, although methods have been developed that allow the use of solids. In general, the quality and rate of GFAAS analysis is dependent upon the success of sample preparation procedures. Sample introduction involves the transfer of a standard or prepared sample into the graphite furnace, and the method employed depends on the state of the sample after
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sample preparation. Due to problems with this process, Browner and Boom [4] has referred to sample introduction as the "Achilles' Heel of Atomic Spectroscopy." These two processes are closely related and hence will be considered together in this section. A book edited by Sneddon [5] discusses various methods of sample introduction in atomic spectroscopy. Liquid, gaseous, and solid samples are all determined by graphite ftwnace atomic absorption, and here a general overview of sample preparation/introduction will be provided, along with representative applications. A detailed discussion on sample preparation as it relates to clinical and biological samples is presented later. Conventional dissolution methods (acid digestion, combustion, fusion) as well as methods to analyze solids with minimal sample preparation (slurry and solid sampling) are discussed. Methods of preconcentration/isolation of analyte, such as extraction, chromatography, and flow injection, allow the removal of the analyte from its matrix and a reduction in the detection limit. A variety of applications of these methods have been employed with GFAAS. GFAAS has also been used to obtain quantitative information on the chemical form of metal present in samples, which is called metal speciation.
1.6.1 Liquids Aqueous samples (e.g., fiver water, sea water, etc.) can be introduced directly into the graphite ~ a c e with an autosampler. If the sample is viscous, such as blood, or colloidal (milk), then it is necessary or advisable to dilute the sample with an appropriate solvent. Usually deionized water or dilute nitric acid are employed for this purpose. Surfactants, such as Triton X-100, are added to some samples to lower surface tension and promote thorough mixing of the diluted sample. The use of a digestion procedure has been shown by some analysts to improve the detection limit and remove some interferences. The determination of lead in blood has been widely investigated due to the toxicity of the element, the relatively low concentration levels (typically 1 ng/mL in "normal" blood), and severe matrix effects. Deval and Sneddon [6] described a method for the direct, simultaneous determination of cadmium and lead in blood reference samples with self-reversal background correction. The use of an ammonium dihydrogen phosphate chemical modifier allowed the use of an elevated pyrolysis temperature that removed the blood matrix. A detection limit of 1.06 ng/mL was reported for lead, which allowed its direct determination at concentration levels between 4.8 and 17 ng/mL. Good accuracy was obtained by this method.
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1.6.2 Solids The majority of samples for analysis by GFAAS are solids, which are generally converted to solutions, and introduced in that form into the graphite tube for analysis. Some solids may be dissolved by simple dissolution in water (e.g., heroin) but most require a digestion procedure. Most procedures involve the dissolution of 0.1-1 g of solid per 100 mL solution. The primary types of dissolution procedures are wet decomposition (acid digestion), combustion (dry ashing), and alkali fusion. The direct analysis of solids is also possible, and two basic techniques have been employed: slurry sampling, in which a powdered material is suspended in a solution that is aspirated into the atom cell (see chapter 6 by Matusiwiecz), and solid sampling, in which a solid is directly inserted into the graphite furnace. Sample preparation methods are generally considered to be critical to quantitative analysis became significant errors may occur due to loss of analyte due to volatilization or precipitation The use of standardized methods of sample preparation would facilitate meaningful comparison of detection limits, linear dynamic ranges, and other analytical figures of merit between various spectrometers. 1.6.3 Wet Decomposition Wet decomposition, or acid digestion, involves the use of mineral acids and oxidizing agents (hydrogen peroxide) to affect dissolution of a sample. Acid digestion is employed for a variety of organic and inorganic solids. Wet digestion procedures may be used to dissolve the entire sample (total decomposition), dissolve a fraction of the entire sample (strong attack), or simulate the transfer of elements in the environment, such as the assimilation of elemems from soil by plants (moderate attack). Acids commonly used in these procedures include nitric, sulfi~c, perchlofic, hydrochloric, and hydrofluoric. Hydrochloric acid is usually not recommended for ftLmace analysis to avoid chloride interferences Nitric acid generally serves as the primary oxidizing acid, and sulfuric acid is a dehydrating agem and has a high boiling point (300~ which increases the rate of decomposition of some samples. The combination of hydrogen peroxide with sulftndc acid produces permono sulfuric acid in-situ. Perchloric acid, although a potential explosion hazard, is a very strong oxidizing agent, and hence is typically mixed with nitric acid to reduce its reactivity. Hydrofluoric acid is required for the dissolution of silicates. Total decomposition of most samples requires hydrofluoric acid combined with other acids. Strong attacks, which are performed with strong acids without hydrofluoric, are easier to use, but will not dissolve silicate residues. This selectivity may be an advantage if the goal is to evaluate levels of pollution.
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Moderate attacks typically involve treating samples with dilute acids or other salts to evaluate the bioavailability of elements. In other applications, it is desirable to monitor the concentration of a metal that is exchanged by a cation of an added salt solution (e.g., ammonium acetate, potassium chloride), which is called the exchangeable concentration. Wet decomposition can be performed with either open or closed systems. Open systems may include teflon beakers, or test tubes in a shallow aluminum block, on a hot plate. Open acid digestion is suitable for relatively "easy" samples (e.g., food and agricultural samples) and is relatively inexpensive, but is unsuitable for some samples, relatively time-consuming (1-24 hours or more), and may allow evaporative loss of volatile elements. Closed digestion systems allow pressures above atmospheric to be developed in the vessel. Higher pressures allow the acids to boil at higher temperatures, and facilitate complete oxidation of the sample. The efficiency of digestion is commonly evaluated by the residual carbon content, which is a convenient, quantitative measure. In addition, loss of volatile elements is eliminated and the rate of digestion is increased. Examples of closed digestion systems include a decomposition bomb, high pressure asher, or a microwave digestion vessel. The former consists of a teflon container surrounded by a stainless steel body. After introduction of the sample and reagems, the emire bomb is heated in a muffle fiamace at temperatures up to 200~ Higher temperatures may be achieved with a high pressure asher (HPA) system, which is composed of a quartz digestion vessel mounted in an autoclave. Unlike the decomposition bomb, this system allows simultaneous monitoring of the temperature and pressure of the sample during the decomposition procedure. Several sizes of vessels are available (2-70 mL); the smallest fits directly on a Perkin-Elmer GFAAS autosampler, allowing analysis from the digestion vial. Microwave digestion involves the use of 2450 MHz electromagnetic radiation to dissolve samples in a teflon or quartz container. Microwaves interact with polar molecules and induce alignment of the molecular dipole momem with the microwave electric field. The field changes constantly, causing rotation of the molecules and intermolecular collisions, producing heat. Consequently, the rate of microwave digestion is dependent upon the coupling efficiency of microwaves with mineral acids. Nitric acid has the highest efficiency, with a value nearly as high as water, followed by hydrofluoric and sulfuric acids. Microwave ovens specific for chemical digestions are recommended for safety considerations. Both open and closed systems have been used with microwave digestion. Most dissolutions are performed with teflon vessels because it is inert with respect to metals, although the maximum temperature to which they may be heated is 200~ This property prevems the use of large quantities of
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sulfuric acid (boiling point 300~ which may deform the vessels. To obtain higher temperatures, quartz vessels are employed. Closed systems allow faster digestions (< 30 minutes), the digestion of more difficult samples (e.g., polymers, geochemical), and a reduced risk of analyte volatilization, but are relatively expensive. For example, a commercial microwave oven is $15,000-$20,000, and digestion vessels are approximately $100 each. A particular advantage of microwave dissolution procedures is the ease of automation. A relatively new development is the combination of microwave digestion with on-line sample and reagent flow transport. These commercial instruments (CEM, Matthews, North Carolina and Questron, Mercerville, New Jersey) offer the potential for rapid, automated sample preparation. Solid samples are converted to 0.1-1% slurries by the addition of suitable acids. As with slurry sampling techniques discussed briefly below (see Chapter 6 for greater details), it is usually necessary to produce samples with a homogeneous, small particle size to ensure a uniform slurry. Some samples may require a "pre-digestion" step in order to prevent plugging of the instrument. Agitation of the slurries is performed by a paddle or ultrasound in order to produce a homogeneous slurry. An aliquot of slurry is obtained in a sampling loop, and then pumped through the microwave system. An output autosampler is used to control the volume of digest delivered. Mineral acid solutions are placed in an acid-rinse reservoir to facilitate selfcleaning of the instrument. In our opinion, the relatively high cost and moderate performance of these instruments makes their value questionable for GFAAS. In addition, it is necessary to homogenize and size-fractionate samples, as required with slurry sampling GFAAS. Slurry sampling accessories are more economical than continuous-flow digestion systems, and analysis can be performed directly after slurry formation. 1.6.4 Combustion
Combustion (dry ashing) procedures involve heating a sample to a sufficiently high temperature (400~176 to remove the organic constituents. The traditional method involves placing the sample in a crucible (platinum or ceramic) or a test tube, followed by insertion in a muffle fiarnace for 1-6 hours to induces quantitative decomposition and removal of organic material. The residue is composed of carbonates and oxides. The analyte is then extracted from the ash with a mineral acid (usually nitric acid for furnace work). Dry ashing has the advantage of relative ease of use and allows decomposition of relatively large sample sizes followed by concentration in a relatively small volume of acid. However, it is a relatively slow process and is limited to relatively "easy" samples. Losses of volatile elements (Hg, As, Se, Cd,
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Pd, T1) may occur. Losses of analyte may also occur due to retention of analyte in the ash. For example, nitric acid will not dissolve silica present in the ash. In general, combustion methods have been replaced by wet decomposition procedures for most analyses by GFAAS.
1.6.5 Fusion Fusion procedures are well suited for the dissolution of samples that cannot be dissolved by other procedures (e.g., geological samples). The sample is mixed with a four-to-ten-fold excess of a fusion reagent, which are usually alkali metal hydroxides, carbonates, or borates (e.g., lithium metaborate), and placed in a platinum or graphite crucible. The crucible is then inserted into a muffle fin'nace at 800-1000~ for 15 minutes - 6 hours to form a molten salt. The melt is then poured in a dilute acid solution (usually nitric acid for GFAAS). The principal advantage of fusion is its applicability to nearly all samples. On the other hand, the large quantities of flux reagents may increase the blank level, making the technique unsuitable for many GFAAS analyses. In addition, volatile elements may be lost in the fusion step.
1.6.6 Solids analysis with slurry sampling (see Chapter 5) An alternative to the dissolution of powdered samples is a technique called slurry sampling, in which the material is suspended in a liquid diluent. The liquid depends on the nature of the sample. For example, for most biological and agricultural samples, the diluent is usually dilute (5 %) nitric acid with a surfactant (e.g., Triton X-100) to ensure good wetting of the sample and to prevent the formation of clumps. The elimination of a dissolution step has the advantages of reducing analysis time and the probability of analyte loss during sample preparation. In addition, quantities of reagents are frequently decreased, which reduces the risk of contamination, and less sample dilution isrequired, which may lower the determinable mass of analyte. GFAAS is well suited to slurry sampling compared to flame and plasma methods because of the relatively long time that the sample remains in the atomizer (long residence times) which usually induces complete atomization of particles. In order to obtain precise and accurate results by slurry techniques, it is necessary to produce a homogeneous slurry. This usually requires the use of a mill to convert the sample into a powder with a small (< 10 ~tm), homogeneous sample size. Typically 1-15 mg of powder are added per 5 mL of diluent. An effective method of agitating the slurry is also required. Methods of agitation include the stabilization of the slurry with a thickening agent (e.g., glycerol) or homogenization of the slun'y. Stabilization can prevent accurate pipetting by the autosampler and hence is not recommended.
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Homogenization has been performed by use of a magnetic stir bar, an vortex mixer, the introduction of gas bubbles, high pressure homogenization, or an ultrasonic probe. Magnetic stirring has the disadvantage that magnetic particles may adhere to the bar. Vortex mixing has been shown to be ineffective at producing homogeneous slurries of dense materials (e.g., sediments) and cannot be easily automated. The use of manual pipetting is inconvenient and also results in a degradation in precision. Gas bubbling was shown to ineffective at forming homogeneous slurries of samples in which the analyte was associated with larger particles. Ultrasonic agitation appears to be the method of choice for slurry preparation. Ultrasound induces disaggregation, wetting, and dispersal of solid particles in a liquid. In addition, it has been shown to enhance extraction of analyte into the diluent. This commercially-available device (Perkin-Elmer, Norwalk, Connecticut) consists of a titanium ultrasonic probe mounted above the autosampler that effectively agitates powdered samples. A gas-actuated cylinder is employed to control the vertical position of the probe, and its operation is synchronized with the autosampler. After the sample has moved directly below the autosampler arm, the probe moves into the sample cup, and the ultrasound is activated to produce a homogeneous sample. The probe is then lifted out of the sample and the ultrasound turned off. The autosampler arm then enters the cup, removes an aliquot, and dispenses it to the fiwnace. Slurry sampling has the potential for rapid analysis compared to dissolution procedures, but with some limitations. First, the measurement of small masses of sample (2 - 50 mg) is required, which is time-consuming, and may not be representative of the bulk sample. Second, it is usually necessary to characterize the particle size and homogeneity of the sample, as well as the distribution of the analyte between the solid and liquid phases of the slurry. If a significant fraction of the analyte is extracted into the diluent, the analytical performance will be similar to a digestion. However, if the analyte remains associated with the solid, then the precision will probably be reduced compared to digestion procedures. Third, careful optimization must be performed to obtain good results. GFAAS parameters to be considered include pyrolysis and atomization temperatures, amount and type of chemical modifier, and oxygen ashing. In addition, it is also necessary to characterize the sample in terms of homogeneity, density, and particle size. It is generally assumed that at least 50 particles should be introduced in each 20 laL injection into the graphite tube. For a material with a density of 1 g/mL, 20 mg of sample is required per milliliter of diluent. Although accurate analyses have been performed with particle sizes exceeding 100 ~tm, it may be necessary to use a nonstandard autosampler capillary. The precision may be degraded as well.
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1.6.7 Direct solid sampling Direct introduction of solid samples (direct solid sampling) eliminates sample preparation procedures, which reduces analysis time and prevents contamination by reagents. In addition, there is no dilution of the sample, which allows measurement of lower levels of analyte than dissolution procedures. Typically a few milligrams of solid material are introduced into the furnace. 1.6.8 Laser ablation Laser ablation (LA) involves the use of a laser beam to ablate or vaporize a solid sample. Spectroscopy may be done in the plasma generated by the laser beam (see Chapter 7 on Laser Induced Breakdown Spectrometry (LIBS)), or the vaporized sample may transported to a conventional atom cell. The majority of work in this area involves the use of an inductively coupled plasma (ICP) as the atom cell, with detection by either optical emission spectrometry (OES) or mass spectrometry (MS). The popularity of LA-ICP methods is due to the low transport efficiency (-~ 1 % ) of conventional nebulization methods of sample introduction. Laser ablation allows a significant improvement in efficiency. Of course, the efficiency of GFAAS is 100 %, and hence no gain in efficiency will be achieved by LA. ICP-OES and ICP-MS also have the advantage of being multielemental techniques, compared to GFAAS, which until the early 1990s was almost exclusively single-elemental. However, laser ablation has the ability to vaporize microparticles (e.g., individual crystal grains in minerals), which cannot be achieved with other solid sampling techniques. LA-GFAAS has considerable potential for microsampling, although more development is required to develop standard accessories and methods. Commercial ablation cells are available for ICP techniques which can be used for GFAAS. One problem remains the relatively low collection efficiency of metals by impaction methods. The use of electrostatic precipitation may be useful in this regard. 1.6.9 Pre-concentration/separation methods The levels of elements in some samples (e.g., semiconductor industry, environmental samples) are below the detection limits attainable by graphite fim~ce atomic absorption. Some matrices (e.g., silicates) cause significant degradation of detection limits, and hence separation of the analyte from the matrix is required. Pre-concentration/separation techniques are used to increase low levels of analyte and remove the sample matrix from the analyte. The enrichment factor (E) is a quantitative measure of the degree of pre-concentration. It is defined as the concentration of analyte after the pre-concentration step divided the analyte concentration in the original solution.
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Commonly used methods for pre-concentration/separation include extraction and chromatography. Major disadvantages of these pre-concentration techniques include their labor-intensive nature and unsuitablity to automation. Recently, flow injection (FI) has been employed with graphite furnace AAS, which has allowed rapid, automated pre-concentration/separation procedures. Other methods include the use of biological organisms for pre-concentration, co-precipitation, solid sorption, and a liquid supported membrane. 1.6.9.1 Extraction
Extraction procedures involve the transfer of the analyte from a solvent (usually water) to a second solvent (usually organic). In order to obtain quantitative extraction, it is necessary to ensure that the analyte is all in the same chemical form, usually as its most common cation, and to control the pH of the aqueous phase. The limit of detection is generally improved by a factor of 10-20 by extraction methods, with a maximum enrichment of approximately one hundred. The analyte must be converted to an uncharged compound or to an ion-association complex in order to increase its solubility in an organic solvent. The extraction process is evaluated in terms of the distribution ratio D : D = C~o,~
(8)
CA,W
where CA,Org and CA,w are the concentrations of analyte in the organic and aqueous phase, respectively. The mass of analyte remaining in the aqueous phase atier n extractions (mA,w,n) is given by
(
tn
Vw mA,w,n -- DVo~g+ Vw mA,w,o
(9)
is the initial mass of analyte in the aqueous phase and Vw and Vo~g are the volumes of the aqueous and organic phase, respectively. In general, it can be assumed that quantitative transfer of analyte may be achieved in one step for D values exceeding 100. Metal chelating agents, such as 8-hydroxyquinoline (oxine) or ammonium pyrollidine dithiocarbamate (APDC), are one class of compounds used to remove analytes from a sample matrix. These chelating agents are commonly weak acids designated by HR. They can be used for a wide variety of metals (Mn+). The equilibrium for the extraction process for a chelate may be expressed as w h e r e mA, w,0
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Mn+ (aq) + nHR (org) ~ MRn (org) + ~
(aq)
383
(10)
and the distribution ratio for a metal-chelate system is given by I!
DM = BnI~x [HRo~g aM [H +]~
(11)
where Kex is the overall extraction constant; g. is the formation constant of the metal chelate; and t3/,M is the fraction of the total metal concentration presem as the uncomplexed metal. Equation (11) demonstrates that the distribution ratio decreases as the pH decreases. Variation of the pH can therefore be used to control the metal ions extracted. The pH,~ value, which is defined as the pH at which 50 % of a metal is extracted, is used to evaluate the selectivity of an extraction. In general, a difference of three units in pH~ value is required to quantitatively separate two metal ions using a single batch extraction. For metals which cannot be separated on the basis of pH, additional complexing agents called masking agents may be employed. Masking agents, which include EDTA and ammonia, serve to tie up one of the metals and prevent its extraction into the organic phase. Ion association complexes involve the formation of a soluble ionic compound containing the analyte and a suitable counterion. In order to be suitable for extraction, these complexes should have no net charge or include sufficient nonpolar functional groups to allow high solubility in nonpolar solvents. Examples of ion-association complexes include Fe(o-phen)32+ / 2CIO4 (where o-phen = orthophenanthroline) and [(C2Hs)20]3IT(H20), / FeCl4. Extraction methods are relatively simple, and may allow extraction of several elemems or only one depending upon the analytical requirements. However, these procedures are difficult to automate, relatively labor-intensive, and have interferences that reduce the extraction efficiency. 1.6.9.2 Chromatography A variety of chromatographic procedures have been employed for preconcentration/separation. Although typical enrichment factors of 100 are obtained, concentration factors up to 2000 have been reported. In order to obtain good accuracy, it is necessary to convert the analyte into one chemical form. A prescribed volume of sample is loaded onto a column using a mobile phase that does not elute the analyte, but (ideally) allows removal of the matrix. A second mobile phase is then added that serves to quantitatively and rapidly remove the analyte from the colmlm, resulting in a relatively concentrated solution.
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Ion-exchange techniques have been employed with cationic resins, which contain acidic functional groups (e.g., -SO3H, -CO2H) and anionic resins, which contain basic functional groups (-NR3H). The exchange processes for cationic resin with a metal Mn+and anionic resin with an anion A n are nRSO3 -H+ (s) + M "+ (a,0 ~ (RSO3")n Mn+ (s) + nil+ (aq)
(12)
and nRYm3 +x" (s) + An" (at0 -'~ (RNR3+)n An" (s) + n X (aq)
(13)
The distribution coefficient for ion exchange (D~) is given by De = concentrationof analyte in the resin (amount / kg dry resin) concentration of analyte in solution (amount / L solution)
(14)
Cationic resins can be used to preconcentrate metal cations, while anionic resins allow the removal of negatively charged interferents and separation of the analyte if an anionic complex of the metal is formed (e.g., ZnCI42). Chelating ion exchange resins, such as Chelex-100, include a functional group that forms chelates with metal cations and have the advantage of forming stronger complexes with most transition metal cations. Chelex-100 and other chelating ion-exchange resins are well suited to sea water samples because they do not interact significantly with alkali metal cations (Na +) which may interfere with conventional ion-exchange resins. A more recent development is the use of preconcentration with a reversed-phase liquid chromatography procedure. A conventional reversed-phase column is used to separate metal ions that have been treated with a chelating agent.
1.6.9.3 Flow injection analysis Flow injection (FI) analysis involves the introduction of a sample (typically 50 btL) in a flowing stream of liquid (~ 1 mL/min) in narrow-bore (0.5 mm), nonwetting tubing for quantitative analysis. A peristaltic pump is generally used to transport the liquid in a laminar flow pattern. A detection system, which is used to measure the analyte concentration, may be virtually any instrument. An autosampler is often used to inject the samples into the flow stream. A variety of types of chemical processes may be automated by the systems. For example, a column, extraction module, or dialysis module may be used to separate the analyte from other sample constituents to minimize interferences in the detection system. Alternatively, reagents may be injected into the system to react with the analyte. It
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is desirable to ensure good mixing by coiling the tubing tightly or packing the tubing with beads to produce a packed bed reactor. The degree of mixing of the sample with the flow stream is referred to as dispersion (D). The dispersion of an FI system is usually quantified by the ratio of the analyte concentration injected (Co) to the analyte concentration at the peak maximum (Cp)
o = Co
(15)
Cp The dispersion of an FI system may be controlled experimentally by variation of several design parameters. For example, dispersion increases with tubing length, tubing diameter, and the flow rate, and decreases with volume injected, tight coiling, and packing with glass beads. As discussed above, the ability of a preconcentration system to increase the analyte concentration may be expressed by the enrichment factor E. However, in some cases a FI system is operated under different experimental conditions than a batch system which may lead to an increase in sensitivity. Consequently, if experimental conditions are not identical between a batch and an FI system, the increase in sensitivity should be referred to as an enhancement factor. Compared to conventional batch procedures, in which each sample is located in a separate vessel (e.g., extraction), FI is a continuous flow technique in which a series of samples are injected into a length of tubing separated by solvent. The basic processes of loading and removal from a column are similar to those employed in chromatography, but FI is distinguished from chromatography because it is designed for rapid quantitative analysis of a limited number of analytes instead of the separation of any number of compounds. FI has been widely employed with flame AAS as a method of preconcentration since the early 1980s became of its compatibility with a continuous flow system. The combination of FI with GFAAS did not occur until the late 1980s, but since then a number of applications have appeared in recent years. The interest in FI- GFAAS may be related to the ability to do automated preconcentration steps and to the availability of a commercially available FI system for use with atomic absorption instnunents. In general, the combination of GFAAS with FI for preconcentration requires specific features in terms of the immanent design First, GFAAS operates in a batch mode, and consequently preconcentration of the analyte is performed in parallel and must be synchronized with the atomization cycle in a discontinuous manner. Second, the maximum volume that can be accommodated in a graphite tube is less than 100 laL; this value is reduced
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with a platform and organic solvems that are commonly used for elution. When preconcentration is achieved by chromatography, it is therefore necessary to use microcolumns (15 laL), and it may not be possible to collect all of the analyte. Third, GFAAS is relatively sensitive to high concentrations of matrix elemems, and hence it is usually necessary to incorporate a column washing step to remove residual sample matrix before elution of the sample. Fourth, the combination of these specifications results in a relatively complicated elution sequence that generally must be computer controlled. Several methods of separation have been employed with GFAAS, including sorption, ion exchange, extraction, coprecipitation, supported liquid membrane, and electrochemistry. Flow injection provides a convenient method for automated sample preconcentration, with typical enrichmem factors of 20-50. We expect this technique to replace batch methods of preconcentration. FI has also been used with GFAAS as an interface for sample introduction into the furnace One interesting application of FI involves its coupling with in situ trapping of volatile hydrides in a graphite tube. The hydride generation (HG) technique involves the conversion of the analyte to a volatile hydride with a chemical reagent (usually sodium borohydride) which is then swept into an atom cell (generally a heated quartz tube) where the molecule dissociates in gaseous atoms. Elements which form volatile hydrides include antimony, arsenic, bismuth, germanium, lead, selenium, tellurium, and tin. Other volatile molecules have been used for sample introduction by similar procedures, including chlorides, fluorides, B-diketonates, and dithiocarbamates. In addition, aqueous mercury may be reduced into the metal which is volatile enough to be determined in a quartz tube maintained at room temperature (cold vapor mercury determination. Disadvantages of these conventional HG procedures include dilution of the analyte by cartier gas and hydrogen and low atomization efficiency in quartz tubes due to their relatively low maximum temperatures. The in situ trapping technique involves flow of hydride into a heated graphite tube which serves to decompose the hydride and condense the analyte on the tube. In general, absolute detection limits are degraded by HG-GFAAS compared to conventional GFAAS, since the efficiency of the HG procedure is not 100 %. The use of flow injection with HG-GFAAS provides a convenient approach to automate sample introduction procedures and a reduction in interferences compared to a batch system. However, in general these methods are relatively difficult to implemem, and the sensitivity for most elements is comparable to that obtained by conventional GFAAS. We consequently do not recommend these procedures for routine analysis.
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1.6.9.4 Other pre-concentration/separation methods Several methods of pre-concentration are available that have been used for a limited number of applications, which include liquid membrane devices, electrochemical cells, co-precipitation, and the use of organisms for preconcentration. 1.6.9.5 Metal speciation Metal speciation is the quantitative determination of each of the chemical forms of a metal presem in a sample. Considerable interest has developed in speciation over the past twenty years because the toxicity and mobility of metals in the environmem and organisms is dependent upon their chemical form. Metal compounds may be classified as inorganic, complexed ions, or organometallic. A variation in the toxicity of differem oxidation states exists for some metals. For example, chromium(VI) is considerably more toxic than chromium(III). In general, the organometallic compounds are more toxic than inorganic compounds because the former have greater permeability through biomembranes and may accumulate in fatty tissues. Mercury is an example of this type of element, where alkylated mercury compounds (e.g., methyl mercury) are more toxic than inorganic mercury (although these species are also regarded as toxic). Tin compounds (e.g., tributyltin) have been of interest because of their use as algicides, fungicides, and molluscicides. These compounds may accumulate to toxic levels in shellfish and fish, although inorganic tin is an essential trace elemem. Arsenic is an exception to the general rule because some organometallic forms, such as arsenobetaine, arsenocholine, and some arsenosugars, are relatively nontoxic, but inorganic arsenic(III) (arsenite) and arsenic(V) (arsenate) are toxic. A considerable body of literature is available on metal speciation. Here we discuss some general aspects of speciation with an emphasis on some recem GFAAS applications. The various chemical forms of a metal must be separated by a method which does not change the chemical structure of the analytes prior to detection by GFAAS or another method. Perhaps the most commonly used separation technique is extraction, either with acids or organic solvents. It is necessary to verify the recovery of the procedure by measurement of the extraction recovery for each analyte. This procedure involves spiking a sample with each analyte and measuring the concentration after extraction. An alternative procedure is derivatization of analytes to achieve preconcentration of the analytes. For example, hydride generation can be employed to preconcentrate hydride-forming elements. Alternatively Grignard reactions may be employed to induce pentylation of alkyllead and alkyltin species and produce compounds that can be separated easily by gas chromatography. Derivatization methods may lead to errors because of
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incompleteness of reactions (e.g., arsenobetaine is not converted to a volatile hydride by sodium borohydride), and probably should be avoided when possible. Some examples of metal speciation with GFAAS include separation of the analytes has been achieved by a number of procedures, including gas chromatography (GC), liquid chromatography (LC), extraction, and coprecipitation A disadvantage of GFAAS with many conventional separation procedures is its incompatibility with a flowing system. These problems may be alleviated by performing the separations in parallel by flow injection methods. Although clearly more work needs to be done in this area, quantitative direct speciation by GFAAS without any separation steps would certainly reduce analysis time and complexity. In conclusion, although traditional methods of speciation may be difficult to interface with GFAAS, flow injection provides a convenient way to determine various forms of elements in an on-line, automated fashion. We expect a number of new methods to be developed in this area. 1.7 Determination of elements by GFAAS One of the goals of this chapter is to provide some guidelines for quantitative analysis by GFAAS. First, the criteria that are used to evaluate whether GFAAS can be used to do a particular analysis are outlined. The second section discusses sampling, storage of samples, and sample preparation. The emphasis is on contamination, which is a common source of error in trace analysis. The use of quality control procedures is discussed to evaluate analytical procedures, including the use of standard reference materials and recovery checks. The instrument optimization protocols required to do quantitative analysis by GFAAS are discussed, such as pyrolysis temperature optimization, atomization temperature optimization, and the type and quantity of chemical modifier. 1.7.1 Applicability The applicability of an elemental analysis technique involves consideration of the analyte, the amount of available sample, and the concentration levels of the analyte. The first criterion involves consideration of the applicability of GFAAS to determine a particular elemem. In general, GFAAS is applicable to the determination of most metals and metalloids, with the exception of a few refractory elements (e.g., tantalum). The atomic absorption cookbook given by all commercial instrtnnents provides a list of determinable elements. The amount of sample must also be considered. An advantage of GFAAS compared to other techniques is the small amount of sample required. Each graphite fiu~ace cycle employs approximately 10-20 pL of solution (dissolved sample or liquid), and since determinations are normally performed in triplicate, approximately 30-60 pL are required. If a smaller volume is available, but the
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analyte levels are relatively high, it is possible to dilute the sample before analysis to increase the volume. Slurry and solid sampling methods may employ as little as 1-4 mg of solid per cycle, allowing analysis of a few milligrams of sample, although such small masses may not be representative of the bulk of the material. Assuming the analyte is determinable by GFAAS, and sufficient sample is available, the third criterion to consider is the concentration levels in the sample following sample preparation procedures (See 1.6). Generally 0.1 - 1 g of solid sample are dissolved and diluted per 100 mL volume. The useful (linear) range of the calibration graph is usually assumed to be between the limit of quantitation (approximately five times the detection limit) to the level of linearity, typically two to three orders of magnitude. It may be possible to detect values closer to the detection limit, but degraded precision and accuracy should be expected. In addition, some sample matrices may degrade the detection limit, increasing the limit of quantitation. Data from the atomic absorption cookbook and the approximate concentration of analyte in the sample should be used to determine whether the levels fall within the useful range of the graph. Obviously, if this condition is met, the analyst may continue to the next step. However, if the concentration levels are too low, the analyst has two options. The easiest option, if available, is the use of a more sensitive technique. This may not be possible became GFAAS is one of the most sensitive techniques. Possible options include inductively coupled plasma- mass spectrometry (ICP-MS) and neutron activation analysis. The second option is to use one of the preconcentration techniques (e.g., extraction, chromatography, flow injection). These techniques also offer the advantage of separating the analyte from the matrix, which may reduce interferences. Their primary disadvantages are their time-consuming nature and inconvenience. If the concentration levels are above the useful range of the calibration curve, several options are available. First, if the concentration levels are sufficiently high, the use of a less sensitive technique, such as flame AAS or inductively coupled plasma optical emission spectrometry, is appropriate. These techniques are faster and usually easier than GFAAS. A second option is dilution of the sample by deionized-distilled water, which has the advantage of diluting potential interferences. Third, many elements have less-sensitive alternative wavelengths, listed in the cookbooks, that may be employed to determine relatively high concentration levels. A final option is the use of a low internal flow of gas through the graphite tube during the atomization step, which serves to more quickly remove the atoms from the atomizer, and reduce the sensitivity. This option should probably be used as a last resort because a gas flow may reduce the temperature during the atomization step and cause chemical interferences.
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The concentrations of metals or elements in many biological and clinical samples are low, and ot~en very little sample is available. The electrothermal atomizer or graphite fiamace also allows in-situ treatment of the sample such as removal of potential interfering and often complex matrix. Hence, the attractiveness of GFAAS in the biological and clinical area.
1.7.2 Sampling, sample storage, and sample preparation The use of well-designed sample collection and storage procedures is required to ensure collection of representative samples with good precision and accuracy. In order to obtain a representative sample, it is first necessary to consider the size of the gross sample required that is truly representative of the entire sample. It is then necessary to reduce the gross sample to laboratory samples, which are employed for analysis, with maintenance of the chemical integrity of the analytes. Ideally, in addition to ensuring that the laboratory sample is representative of the entire sample, it is necessary to ensure that no addition or deletion of analyte has occurred prior to analysis. Reduction of the sample size involves homogenization of the sample by thorough mixing. Some samples, such as soils and fertilizer blends, are heterogeneous. In these cases the particle size should be reduced as much as possible in order to obtain representative portions for analysis. Particle size reduction of hard materials may be performed with laboratory mills and grinders. Sot~ samples, such as foods and tissue, may be homogenized with mixers or blenders. It should be pointed out that in some cases it may not be desirable to homogenize the entire sample. For example, consider fruits with non-edible skins The levels of metals may be higher in the skins than in the fruit due to pollution. However, is the concentration in the skin of interest? It may be more appropriate to analyze the edible portion. Loss of analyte may occur during transport, storage, and sample preparation. For example, the analyte may coprecipitate with other salts (e.g., urine samples). These losses may be eliminated by complete digestion of the sample. The analyte may absorb on to the wall of a container. Absorption losses can be minimized by the use of thoroughly cleaned Teflon or polyethylene containers, acidification of the sample to pH < 1, and minimization of the contact time. Although volatilization of elements is normally associated with sample preparation procedures, losses of volatile compounds (e.g., mercury compounds) may occur at room temperature. Contamination is a particularly significant factor in GFAAS because of the relatively low (pg/mL - ng/mL) concentration levels determined. Air particulate matter (dust) may be a major source of contamination, both at the sampling location and in the laboratory. When collecting plant samples, it is ot~en desirable to
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monitor the concentration of metals in tissues independent of the air particulate matter. In this case it is necessary to wash the samples in order to remove the deposition, which may lead to additional contamination/losses. The prevention of contamination by air particulate matter may be achieved by the use of clean environments. Class 100 clean rooms appear to be adequate at removing most particulates. Ideally, the air entering the clean room should be purified with high efficiency particulate air (HEPA) filters, which serve to remove at least 99.99 % of 0.3 l~m particles. The analyst may also be a source of contamination. Human skin is a source of sodium and other elements. Significant contamination may also be produced by hair and clothing. Ii is recommended limiting access to the clean room and requiring special dust-~ee clothes, shoes, and hats. Equipment and chemicals are potential sources of contamination. Collection and storage should be performed using clean containers (washed in detergent followed by soaking in 1 M nitric acid) made of a high-purity material in a relatively clean area with controlled temperature conditions appropriate for the samples. For trace element analysis, recommended materials include polyethylene, teflon, and synthetic quartz. Sample vessels should be permanently labelled to allow random assigmnent to prevent bias from particular containers. Colorless pipette tips should be used for solution preparation because the color of some tips is due to the presence of certain metals. Homogenization of samples by grinding or blending induces considerable physical contact between the sample and this equipment. Considerable contamination may be induced during this step in the sample preparation procedure. The choice of material to be used for grinding is dependent upon the analytes. For example, steel is a durable, relatively inexpensive material, but may induce contamination of iron, chromium, and manganese, while tungsten carbide is brittle and expensive but elemental contamination is limited to tungsten, cobalt, and a few rare earth elements. High quality deionized water is essential for trace element analysis. A number of water purification systems are available commercially. Further purification of water using a sub-boiling distillation unit may be necessary for extremely low concentration levels. The use of high purity chemical reagents is obvious for trace analysis. Contamination may be a problem even with the use of high quality reagents. The levels of copper and zinc in blood samples are typically in the ~tg/mL range, and contamination from acids is not a problem. However, the other elements are present at the pg/mL- ng/mL levels, and concentrations in the reagents are the same as or greater than those in the samples. These data indicate that it may be necessary for the laboratory to purify acids for trace analysis. Quartz sub-boiling
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distillation units may be used to reduce metal concentrations in all acids except hydrofluoric, which must be purified in all teflon units. Details on sample preparation procedures are given in section 1.6. The significance of contamination control is given in this example, where the determination of manganese in serum by neutron activation analysis is reported in the absence and presence of contamination controls. Contamination controls for this work involved the use of care in sample handling, carefully cleaned glassware and plasticware for the collection and storage of samples, purified reagents, and a cleanroom. The concentration levels reported here are of the same order of magnitude as would be expected by GFAAS, and similar results would be expected with this technique. The manganese levels are a factor of ten lower in the absence of contamination, and the relative standard deviation (RSD) was reduced from approximately 100 % to 15 %. These data illustrate the concept of "garbage in, garbage out" for elememal analysis. Reliable data cannot be obtained during the analysis step if errors are introduced in the collection and preparation steps. This trend has been confirmed in several analyses in the environmental (e.g., sea water) and clinical chemistry (e.g., blood serum) literature, in which ambient levels of metals have "fallen" over the past 30 years because of the elimination of contamination in more recent work.
1.7.3 Quality control procedures Quality in analytical procedures is characterized by the magnitude of errors and the extent to which the errors affect the final results. Accredited laboratories are required to document the accuracy and precision of methods and results as described by international organizations such as the International Standards Organization (ISO), International Union of Pure and Applied Chemistry (IUPAC), and Association of Official Analytical Chemists (AOAC). All samples and procedures must be carefully documented during collection, storage, and analysis. Careful attention must be paid to blanks and calibration standards. Calibration and reagent blanks should be prepared and analyzed to establish a zero baseline and a background value, respectively. Generally two independent sets of high quality calibration standards should be employed. Calibration standards are generally prepared by serial dilution of concentrated stock solutions (1000 ~tg/mL). Directions for preparation of stock solutions are provided with the list of standard methods provided with the instrumem, or alternatively commercial standards may be purchased. Stock and calibration standards are usually stored in acid solution in plasticware to increase their stability. Analytical standards for GFAAS should be prepared daily in plasticware (rather than glassware) in dilute nitric acid (0.2 %) by serial dilution techniques. Generally dilutions should be performed with mechanical pipettes with volumes between 0.1 and 5 mL.
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Standard reference materials (SRMs), which are samples that have been analyzed by at least two independent methods, should be analyzed with the "real" samples to assess accuracy. A wide variety of standard reference materials are available, such as manufactured matrix (e.g., plant process quality control for metals, cement, glass, paint, and automobile catalysis), agricultural products, environmental samples (soil, sediment, sludge, and water), botany, marine science, geology, coal, and medicine. Some government agencies that supply SRMs are listed in Table 1. A number of private companies also produce reference materials. Recovery checks involve the addition of an aliquot of analyte to a "real" sample to evaluate the recovery efficiency of the method. Recovery checks should be incorporated randomly in a sequence of analyzed samples. The level of spiked analyte may be equal to the expected level of analyte, in order to evaluate differences between analyte from the sample or from the spike. On the other hand, minimal error in the recovery factor is obtained when the added analyte is several times larger than the native analyte. It is necessary to ensure that the total concentration of analyte is on the linear portion of the calibration graph. Samples should be analyzed using strict quality control procedures. Blind samples are standards that are submitted for analysis as "real" samples. Analysis of replicates involves repeated analysis of a sample during a series of measurements in order to evaluate the precision of the analytical system. Typically at least one sample should be replicated in every ten analyses. In addition to quality control within a laboratory, it is also necessary to verify comparability between laboratories. External quality assurance systems are employed to assess the reliability of results from more than one laboratory. 1.7.4 Development of GFAAS methods The successful use of GFAAS for real sample analysis involves the use of modem fia'nace technology [1, 2]. Method development should be initiated by consultation with an atomic absorption "cookbook" of experimental conditions for the determination of a particular element, provided by most manufacturers. Methods are usually outlined for the determination of elements in particular samples which may include the concentration levels that correspond to the linear region of the calibration graph; sample preparation procedures; immanent conditions, such as available wavelengths, with their relative sensitivities, slitwidth, and temperature programs; and type and amount of chemical modifiers. However, it is the opinion of the authors that the analyst should use these conditions as general guidelines and develop their own sample preparation methods and instrument conditions. Additional information may be obtained by careful examination of the atomic spectrometry literature before attempting an analysis to avoid "reinventing the wheel." Methods of sample preparation are discussed in
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section 1.6. It is recommended that the reader focus on references published since the mid-1980s, subsequent to the development of modem furnace technology, for the most relevant information. Table 1 Representative government suppliers of standard reference materials
Agency Community Bureau of Reference (BCR), Brussels, Belgium (numerous standards in many areas) National Institute of Standards & Technology (NIST), Gaithersberg, Maryland, USA (numerous standards in many areas) National Research Council (NRC), Montreal Road, Ottawa, Canada (marine standards) National Institute for Environmental Studies (NIES), Ibaraki, Japan (environmental standards) Geological Survey of Japan, Ibaraki, Japan (geological standards) United States Geological Survey (USGS), Denver, Colorado, USA (geological standards) Laboratory of the Government Chemist (LGC), Middlesex, United Kingdom (numerous standards in many areas) Canadian Certified Reference Materials Program (CCRMP), Ottawa, Ontario, Canada (geological standards) Agricultural Research Center (ARC), Jokionen, Finland (agricultural standards) National Research Center fro certified Material (NRCCRM), Beijing, China (numerous standards in many areas) ,
,
A number of GFAAS instrumental conditions need to be selected or optimized. For example, a method of background correction should be selected, if more than one is available on an instrumem. Many modem instrumems have selfreversal or Zeeman and the continuum source method. In general, self-reversal or Zeeman is preferable to the cominuum source method, although there are exceptions to this statemem.
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The type of graphite must be selected as well. A pyrolytically coated graphite tube with platform atomization gives optimum performance for most elements. However, if cost of graphite is an important consideration, some volatile elements may be accurately determined with less expensive uncoated graphite tubes, and some involatile elements may be accurately determined without a platform. The cookbook should be consulted for the type of graphite recommended for a particular application. The use of chemical modifiers has been shown to reduce chemical interferences for GFAAS. The atomic absorption cookbook and literature should be investigated for the most appropriate choice of chemical modifer for a given analyte. For example, palladium has been commonly used to determine a variety of volatile elements because of its ability to stabilize them sufficiently to allow pyrolysis temperatures above 1000~ and remove much of the matrix. Frequently several modifiers have been employed for a given element, and it is desirable to experimentally evaluate the most suitable. After the modifier has been selected, it is necessary to optimize the amount of reagent employed. Chemical interferences caused by organic matrices have been effectively removed by the use of oxygen ashing at temperatures below 800~ to prevent oxidation of the tube. Lastly, the atomization cycle of the graphite tube needs to optimized for the particular analysis. Most cookbooks provide temperature programs that can be used as a starting point. In general, conditions for the dry cycle are determined by the graphite employed (e.g., whether a platform is present or not), and hence they usually do not need to optimized for each analysis. The pyrolysis and atomization temperatures are optimized for aqueous standards and the samples. Ideally, no difference would be observed in the optimum temperatures for standards and samples. The characteristic mass or limit of detection should be determined and compared to values specified in the cookbook to evaluate the analytical performance of the system. Quality control procedures should then be employed to evaluate the precision and accuracy of the analysis. These procedures include the sample collection methods, including contamination control, the use of high quality standards, and the use of standard reference materials and recovery checks. From the discussion above, it should be apparent that a number of experiments must be performed in order to optimize conditions and perform a determination for elements or metals by GFAAS. Normally one might expect to spend a few hours to several days performing optimizations for the determination of an element in a "new" sample.
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1.8. APPLICATIONS The final part of this chapter describes recent developments in instnnnentation, methodology, and selected applications for GFAAS. It is not designed to be a comprehensive review, but to describe some of the more interesting developments in these techniques. Recently, Butcher [7] has reviewed some of the more interesting and innovative areas of GFAAS. Some recent reviews on the application of GFAAS to the biological and clinical area should be consulted [8, 9, 10, l l] 1.8.1 M u l t i e l e m e n t continuum source G F A A S
One of the traditional weaknesses of ETA-AAS is its low sample throughput because of single-element detection. In recent years, several instruments have been developed that involve the use of multiple hollow cathode lamps to allow simultaneous determination of 2-6 elements [12]. However, these instruments require lamp changes and optical recalibration for a different set of elements. An alternative instrument design to HCL excited ETA-AAS, using a continuum source (xenon short-arc lamp) using an echelle spectrometer with a two-dimensional charge-injection device (CID) array detector has been developed [13]. This system provides greater simplicity with the high spectral resolution required for continuum source ETA-AAS. The analytical capabilities of the instrmnent were evaluated by the determination of cobalt, nickel, copper, zinc, and lead. Table 2 summarizes detection limits of this instrument compared to the detection limits of manufacturer's HCL-excited system using the electrothermal atomizer. In all cases, the continuum source system had higher values than the commercial system. These data also demonstrate that the detection limits degrade as a function of wavelength. This was attributed to a reduction in the intensity of the continuum source below 280 nm. In addition, the detection limit for copper degraded by a factor of three in the multi-element mode compared to the single-element mode. This degradation was attributed to an increase in integration time and background scatter in multi-element analysis. The ability of the system to perform real-sample analysis was verified by the accurate determination of lead in drinking water. In summary, this prototype insmunent has considerable potential for multielemental analysis, although the system is limited by relatively poor detection limits because of the low intensity of the continuum source in the ultraviolet. The authors suggested the use of a pulsed continuum source would provide better intensity in this region of the specmnn, resulting in better detection limits for many elements.
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Table 2 Comparison of detection limits for Echelle/CID system to the manufacturer's detection limit Element
Wavelength nm
Cu
324.75
Pb Co Ni Zn
283.31 240.73 232.00 213.86
Echelle/CID Detection Limit, pg 3 9 8 90 60 30
Elements Manufacturer" s Simultaneously Detection Limit Determined HCL-AAS, pg 1 0.5 3 3 4 3 8 3 5 3 1
1.8.2 Determination of lead in blood by tungsten-coil AAS The determination of lead in blood is a very common analysis because of the toxicity of this metal, particularly to children [ 1]. Although a variety of techniques have been proposed for this analysis, ETA-AAS has been widely used because of its excellent detection limits in the low picogram range and its use of small sample volumes. The use of a chemical modifier, such as palladium, allows the use of a sufficiently high pyrolysis temperature to remove the matrix and allow accurate analysis [6]. Salido et al. [14] reported the determination of lead in blood using tungsten coil AAS. A tungsten coil, obtained from a slide projector bulb, fit into a ceramic bulb mount in a quartz cell. Nylon bushings, which contain quartz windows, screwed into both ends of the cell. The cell was purged continuously with 10 %HJAr to minimize oxidation of the coil. A solid state power supply provided 0 15 A at 120 ACV to the coil for temperature control. Non-absorbing lead lines at 280.0 nm and 287.0 nm on either side of the analytical wavelength (283.3 nm) were used to provide background correction. A CCD spectrometer served as the detector. A commercial ETA-AAS spectrometer was used to evaluate the performance of the W-coil AAS instrumentation. Aqueous standards and blood samples were treated by an extraction procedure. A lead complex was formed with ammonium pyrrolidine dithiocarbamate (APDC). This hydrophobic chelate was subsequently extracted into methyl iso-butyl ketone (MIBK). The resulting MIBK solution was then introduced into the AAS instruments. Under optimized pyrolysis conditions with the tungsten coil (2.3 A), acceptable absorbance profiles were obtained. The
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presence of double-humped profiles observed at low pyrolysis temperatures were attributed to incomplete decomposition of the lead chelate. Analytical figures of merit for ETA-AAS and tungsten coil-AAS are shown in Table 3. The detection limits and characteristic masses for the systems were within a factor of two of each other, and the linear dynamic ranges were identical. The method detection limits for both techniques were well below the Centers for Disease Control (CDC) target value of 10 - 20 pg/L. The systems were also compared for the determination of lead in NIST bovine blood SRMs and painters' blood samples. For the SRMs, the tungsten coil instrument results were with 8 % of the certified values with an RSD below 10 %. For the painters' blood samples, the tungsten coil results were 91.7 % of the ETA-AAS values. The tungsten coil system was also shown to meet the CDC required accuracy limits of • 40 pg/L, demonstrating the suitability of this tungsten-coil instrument for the determination of lead in blood. Table 3 Analytical figures of merit for ETA-AAS and ttmgsten coil-AAS
Figure of merit D etecti0n limit Method detection limit Characteristic mass (pg) Linear dynamic range (orders of magnitude)
ETAAS 8 (0.4) 16 (0.8) 13 2
Tungsten Coil-AAS 12 (0.6) 24 (1.2) 28 2
1.8.3 Determination of arsenic and tin Hydride generation [15] (HG) atomic spectrometry involves the chemical conversion of analytes to volatile hydrides which are decomposed to atoms into a suitable atom cell. The atom cell is usually a quartz tube which is heated electrically or inside a flame. For AAS, this technique is particularly sensitive for elements whose absorption lines are below 200 nm, such as selenium and arsenic. A detailed description of HG techniques including GFAAS is provided in Chapter 2 of this book. Pacquette et al. [16] reported the first coupling of HG with laser excited atomic fluorescence spectrometry (LEAFS) for the determination of arsenic and selenium. Laser light from a XeF excimer laser (351 nm) was used to pump a dye laser to produce ~460 nm radiation. A second harmonic generation crystal was used to fi'equency double the visible light to produce ultraviolet light between 230
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and 235 nrrL The ultraviolet light was converted to arsenic and selenium absorption wavelengths (193 - 197 nm) using stimulated Raman scattering for excitation of arsenic and selenium. A laboratory constructed system was used for HG, consisting of a reaction vessel, a glass U-tube water trap (maintained in dry ice-isopropanol to remove water vapor), and a glass U-tube hydride trap (immersed in liquid nitrogen). A commercial ETA served as both the trapping cell and atomizer. Palladium was added to the tube and dried. The graphite tube was heated to 200~ as the hydrides were introduced through a quartz tube in the dosing hole to trap the analyte on the tube. Atomization was performed with a conventional ETA heating program. A comparison of limits of detection of various hydride generation techniques is available in Table 4. Table 4 Limits of detection for arsenic and selenium by selected hydride generation techniques i
HG Technique HG-ICP-LEAFS HG-ETA-LEAFS HG-ICP-OES HG-ICP-MS HG-AAS HG-ETA
Arsenic pg pg/mL 5000 1000 200 40 100 1000 0.6 12 16 80 49 7
i
Selenium pg pg/mL 300 60 800 160 200 20 18 90 29 140 49 7
The HG-ETA-LEAFS detection limits were 200 pg and 800 pg for arsenic and selenium, respectively. These values were approximately 40 times and 400 times worse than the most sensitive HG method, HG-inductively coupled p l a s m a mass spectrometry (ICP-MS), and 1000 times less sensitive than previous ETALEAFS detection limits. The relatively poor detection limits were attributed to low transport/trapping efficiency caused by the laboratory constructed HG system. It was concluded that high efficiencies would lower detection limits to the low picogram or high femtogram mass range.
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1.8.4 Determination of cadmium and zinc by double resonance laser excited atomic fluorescence in an electrothermal atomizer Double resonance (DR) laser-excited atomic fluorescence spectrometry (LEAFS) involves the use of two wavelengths of laser light to promote the analyte atoms to relatively high energy levels with subsequent fluorescence. DR-LEAFS provides high sensitivity, with typically detection limits below 100 fg. In addition, it provides very high spectral selectivity, including excellent discrimination against scattered laser light. Ezer et al. [17] reported the use of DR-LEAFS for the determination of zinc and cadmium. A XeF excimer laser was used to pump two dye lasers at a repetition rate of 30 Hz. For cadmium, the output of one dye laser was frequency doubled to produce 228.802 nm (maximum energy, 540 ~tJ) and the other was used directly (643.847 nm). Cadmium fluorescence was detected at 361.1 or 346.7 nm. For zinc, the first dye laser output was converted to 213.856 by stimulated Raman scattering, and the second used directly at 636.235 nm. The fluorescence was detected at 334.5 nm. Power dependence studies were performed to evaluate whether the transitions were saturated. The cadmium UV transition was saturated at less than 1 ~tJ, but the visible transition was not saturated. The best detection limits were obtained with 361.1 nm detection: 70 fg (7 pg/mL) with the laser tuned on the analytical wavelength (contamination limited) and 40 fg (4 pg/mL) with the laser tuned off the analytical wavelength. In the case of zinc, high background levels of zinc contamination were reported to effect the detection limit. It was also suggested that the contamination could be induced by molecular species, such as NO. The zinc detection limits were 6 pg (600 pg/mL) (on-wavelength) and 700 fg (off-wavelength). The capability of the system to do practical analysis was examined by the analysis of a bovine serum SRM. The sample was diluted in water and analyzed without a matrix modifier. For zinc, a measured value of 940 + 60 ng/g was in good agreement with the certified value of 890 • 60 ng/g. Cadmium could not be determined in the SRM because the levels were below the DR-LEAFS limit of detection. In summary, DR-LEAFS was shown to be a highly sensitive method capable of accurate real-sample analysis. 1.8.5 Copper determination in biological materials by ETAAS using W-Rh permanent modifier A recent study by Lima et al. [18] involves a tungsten-rhodium treatment on the integrated platform of a transversely heated graphite fiimace atomizer was used as a permanent chemical modifier for the determination of copper in biological materials such as copepod homogenate, fish flesh homogenate, tuna homogenate,
Graphite Furnace Atomic Absorption Spectrometry
401
pig kidney, rye grass, brown bread, plankton, and mussel tissue. The samples were all certified by agencies similar to that described in Table 1. The W-Rh permanent modifier was stable up to 250-300 atomization cycles when using volume of 20 OL of a digested samples and increased the lifetime of the graphite tube by over 1000 atomization cycles. Detection limits were in the sub E]g/g range for copper. Accuracy was at least as good as standard GFAAS methods. 1.8.6 Determination of urinary lead, cadmium and nickel in steel production workers by GFAAS The following is an example of the use and value of GFAAS in a clinical situation. A recent paper by Homg et al. [19] describes a GFAAS method for the determination of urinary lead, cadmium and nickel in steel workers. The objective was the screening of workers under routine clinical laboratory conditions. After pre-treatment with acid, the samples were digested via a microwave oven and determined by GFAAS. The analytical reliability (accuracy and precision) of the GFAAS method was ascertained through the certified standards as well as comparison to two electrochemical methods (differential pulse stripping voltammetry and hanging mercury drop electrode) and found to be excellent. This was also confirmed using National Institute Standards & Technology (NIST) (Gaithersberg, Maryland-Standard Reference material (SRM) 2670- freeze dried urine. Typical urine concentrations for lead, cadmium and nickel in steel production workers is shown in Table 5. Also shown in Table 5 are results for quality control (QC) and control concentrations. 1.8.7 Determination of platinum in clinical samples Platinum has been proposed as an anti-cancer drug and its determination in a wide variety of body fluids and tissues is frequently required. In too large a presence or concentration in the body it can be toxic. A recent review by Yang et al. [20] describes various methods, including GFAAS to determine platinum in clinical samples. The high sensitivity of GFAAS makes it attractive for determination of platinum, particularly for patients treated with platinum containing drugs, although a drawback is the complex matrix associated with clinical samples. However, recent pharmo-kinetic studies with oxaplatin suggest that the sensitivity of GFAAS may not be adequate for the accurate determination of the "free" platinum in plasma ultrafiltrate beyond a 24-hr period [21 ]. A GFAAS method was developed for the determination of platinum in human plasma, plasma ultrafiltrate and urine from cancer patients orally receiving a platinum based drug. The sensitivity was enhanced by using a volume of 150-1xL [22]. GFAAS has been used in the determination of platinum in high protein solutions as plasma-protein
J. S N E D D O N and D.J. B U T C H E R
402
bound cisplatin [23], multiple tumour samples [22], neurologic tissue [24], and human plasma. Table 5 Urine concentrations a for lead, cadmium and nickel for steel workers, QC-workers and control groups i
i
lead, ~tg/L 52.3 +19.1 (20.26-89.60)
i
cadmium, ~tg/L i
Production Workers
i
nickel, i
~tg/L i
9.55 + 5.33 (3.08-22.61)
36.6 + 16.5 (17.0-79.5) 29.8 + 13.1 _ (4.45-51.0)
QC-workers
48.0 + 7.9 (37.98-68.61)
7.96 + 2.21 (3.18-10.32)
Controls
31.1 + 16.2 (3.88-58.12)
3.45 + 2.07 (0.60-6.81)
4.39 9 2.35 (1.73-6.82)
Each value represents the mean standard deviation. The number in parenthesis shows the range in each case.
a
1.9. CONCLUSION This chapter has given an overview of GFAAS with selected applications in the biological and clinical area. GFAAS as an analytical technique developed from the late 1960"s until the late 1980"s/early 1990"s by significant improvements in instrumentation (background correction, sample introduction techniques, atomisation techniques (platform, probe or surface)), innovative engineering (constant temperature atomisation, multielement determination, etc.) and a greater understanding of the mechanism of atomization. However, since the early 1990"s it is probably true to say that there have been no significant improvements in GFAAS. This is due, in part, to the fact that the technique has evolved into a standard and widely accepted technique for trace element or metal determination, particularly at the ppb or lower concentrations where there is limited sample available. The ability to treat the sample in-situ is attractive, particularly when there is a complex matrix. The ability to determine a few (two to six elements simultaneously) can be useful although a compromise in experimental conditions often leads to a degradation in detection limit in the simultaneous mode compared
Graphite Furnace Atomic Absorption Spectrometry
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to the single element determination. It will continue to be used to provide necessary and reliable information in many fields, including the clinical and biological fields. REFERENCES 1 D
2e
0
.
5. .
7. 8.
0
10.
11. 12. 13. 14. 15. 16. 17.
D.J. Butcher and J. Sneddon, A Practical Guide to Graphite Furnace Atomic Absorption Spectrometry, Wiley, New York, New York, USA, (1998). K.W. Jackson (Editor), Electrothermal Atomization for Analytical Atomic Spectrometry, John Wiley, Chichester, England (1999). B. Welz, Atomic Absorption Spectrometry, John Wiley, Chichester, England (1998). R.F. Browner and A.W. Boom, Anal. Chem., 56 (1983) 786A. J. Sneddon (Editor), Sample Introduction in Atomic Spectroscopy, Elsevier Science, Amsterdam, The Netherlands, (1990). A. Deval and J. Sneddon, Microchem. J., 52 (1995) 96. D.J. Butcher, Appl. Spectrosc. Rev., 37(3) (2002) in press. S.J. Haswell (Editor), Atomic Absorption Spectrometry: Theory, Design, and Applications, Analytical Spectroscopy Library Ed., Volume 5, Elsevier, Amsterdam, The Netherlands, (1991 ). C. Minoia and S. Caroli (Editors), Applications of Zeeman Graphite Furnace Atomic Absorption Spectrometry in the Chemical Laboratory and Toxicology, Pergammon Press, Oxford, England, (1992). H.G. Seiler, A. Sigel and H. Sigel (Editors), Handbook on Metals in Clinical and Analytical Chemistry, Marcel Dekker, New York, New York, USA, (1994). R.F.M. Herber and M. Stoeppler, Trace Element Analysis in Biological Specimans, Elsevier, Amsterdam, The Netherlands, (1994). K.S. Farah and J. Sneddon, Appl. Spectrosc. Rev., 30 (1995) 351. J.B. True, R.H. Williams and M.B. Demon, Appl. Spectrosc., 53 (1999) 1102. A. Salido, C.L. Sanford and B.T. Jones, Spectrochim. Acta, 54B (1999) 1167. D.L. Tsalev and J. Dedina, Hydride Generation Atomic Absorption Spectrometry, John Wiley, New York, New York, USA (1995). H.L. Paquette, S.A. Elwood, M. Ezer, D.J. Swart and J.B. Simeonsson, Appl. Spectrosc., 54 (2000) 89. M. Ezer, H.L. Paquette and J.B. Simeonsson, Spectrochim. Acta, 54B (1999) 1755.
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E.C. Lima, F. Barbosa, Jnr, F.J. Krug and A. Tavares, Talanta, 57 (1) (2002) 177. C-J. Homg, J-L. Tsai, P-H. Homg, S-C. Lin, S-R. Lin and C-C. Tzeng, Talanta, 56(6) (2002) 1109. Z. Yang, X. Hou and B.T. Jones, Appl. Spectrosc. Rev., 37(1), 2002 57. W. Kern, J. Braess, B. Bottger, C.C. Kaufmann, W. Hiddemann and W. Schleyer, Clin. Cancer Res., 5 (1999) 761. S. Vouillamoz-Lorenz, J. Baur, F. Lejeune and L.A. Decosted, J. Pharm. Biomed. Anal., 25 (2001) 465. T.J. Einhauser, M. Galanski and B.K. Keppler, J. Anal. Atom. Spectrom., 12 (1991)81. M. Kharatishvili, M. Mathieson and N. Farell, Inorg. Chim. Acta, 255 (1997) 1.
INDEX Immobilized borohydride on ion exchange column and moveable reduction bed, 62-64 In-situ trapping/ETAAS, 71-73 Vesicle-assisted, 64-65 Hyphenated techniques, 80 CE/HG, 84-87 HPLC/HG, 80-83
Absorbance, 363-365 Atom formation, 365-366 Atomic absorption spectrometry Applicability, 388-395 Development of method, 393-394 Sampling and storage, 390-392 Quality control procedures, 392-393 Applications, Arsenic and tin, 398-399 Cadmium and zinc by double resonance laser excited AFS in an electrothermal atomizer, 400 Copper in biological materials, 401-402 Lead in blood by tungsten coil AAS, 397398 Lead, cadmium and nickel in urine of steel workers, 401 Platinum in clinical samples, 401-402 Multielement continuum source, 396-397 Flame, 7, 243-244 Furnace, graphite furnace or electrothermal atomization, 7-8, 81-84, 244-246, 361-404 Furnace design and materials, 368-369 Furnace heating cycle, 370-373 Instrumentation, 366-367 Atomic emission spectrometry, 247 Flame, 247 Atomic fluorescence spectrometry, 249, 400 Atomization (methods of), 373-374
Inductively coupled plasma-atomic emission spectrometry (ICP-AES), 8-9, 24-27, 11-13, 8487,247-248 Application, 18-20, Inductively coupled plasma-mass spectrometry (ICP-MS), 27-30, 84-87, 117-120, 248 Applications, 141-149, 201-204, 210-214 Double focusing, 119-120, 125-128 Electrostatic mass analysers, 123 Magnetic mass analysers, 121-123 Nier-Johnson geometries, 124-125 Field -flow fractionation, 179-186 Comparison with SEC, 204-205 Electrical FFF (E1FFF), 192-194, 203 FFF modes, 186-187 FFF Sub techniques, 187-188 Flow FFF (FIFFF), 194-197,203-204 Frit outlet, 216-217 Instrumentation, 198-199 On-channel, 215-216, 221-223 Opposed-flow sample concentration, 217221 Optimization, 199-200 Sedimentation FFF (SdFFF), 188-190, 201-202 Thermal FFF (ThFFF), 190-192, 202-203 Interferences, Atomization in gas phase for HG, 68-71 Chemical in liquid phase and pre-reduction in HG, 74-78 Matrix, 139, Non-spectral, 18 Spectral, 16-18, 129-136 Isotopes, 20-29, 150-159 Accuracy, 150 Blanks, 152-153 Isobaric interferences, 153 Isotope dilution, 157-158 Paleoanthropological, 157 Precision, 153-155 Resolution, 155 Tracers, 20-23, 156-157 Copper, Nickel, 24-26 Calcium, 25-26 Iron, 26-27 Selenium, 27-28
Body fluids or biological or clinical samples (human serum, urine, plasma protein and tissues), 5-7, 15, Direct current plasma, 247 Electron densities, 306-308 Excitation temperature, 304-306 Flow-injection techniques, 246 Hydride generation Applications, 87-114 Arsenic, 87-92 Antimony, 99-102 Bismuth, 107-109 Germanium, 102 Lead, 102 Miscellaneous, 103 Selenium, 92-99 Tin, 102 Electrochemical, 54-55 Fast gas-liquid separation, 60-62
405
Others, 28-29 Laser-induced breakdown spectrometry, 287-290 Analytical characteristics, 293-297 Applications, 309-330 Advanced materials, 328-329 Aerosols and gases, 324-325 Environmental, 309-315 Liquids and solutions, 322-324 Metallurgical, 315-321 Miscellaneous, 329-330 Non-metallic solids, 325-328 Basic principles, 292-293 Factors influencing plasma production, 295306 Ambient conditions, 300-302 Electric and magnetic fields, 302-303 Irradiation energy, 297-298 Physical properties, 298-300 Plasma shielding, 303-304 Sampling geometry, 304 Wavelength, 295-296 Fundamental studies, 290-304 Instrumentation, 314-326 Echelle spectometer, 325-326 Excimer laser, 316 Field instrumentation, 319-322 Fiber based, 316-319 New approaches, 322-325 Nd-YAG laser, 316 Laser-induced plasma production, 293-295
Slurry sampling or slurry sample introduction, 237-284 Calibration, 241 Chemical modification, 241 Nomenclature, 242 Particle size, 240 Precision and accuracy, 242 Slurry concentration, 241 Slurry preparation, 239-240 Speciation, 29-30, 158-159, 205-209, 387-388 Capillary electrophoresis, 169-170 DNA adducts quantification, 165 Gas chromatography, 167-169 HPLC, 159-160 Ion-exchange, 162-164 Off-line strategies, 170 Organics solvents-induced interferences, 167 Selenium, 161-165 Size exclusion, 159 Spectroscopy, 361 Thermal vaporization techniques, 249-250
Microwave-induced plasma-atomic emission spectrometry, 248-249 Reference materials, 30, 394 Reference methods, 30 Sample introduction, 13-14, 374-375 Combustion, 378-379 Chromatography, 383-384 Flow injection analysis, 383-386 Extraction, 382-383 Fusion, 379 Laser ablation, 381 Liquids, 375 Metal speciation, 387-388 Pre-concentration/separation, 381-382 Other, 387 Solids, 357-358,376 Solids with slurry, 379-380 Wet decomposition, 376-378 Sample preparation, 31,378-379 Sensitivity (and limit of detection or detection limit), 139-141,247-251
406