Modern Analytical Chemistry – serie

This book is intended both to be an introduction to techniques and applications of liquid chromatography/mass spectrometry and to serve as a reference for future workers. When we undertook its writing, we chose not to cover the field, particularly applications, exhaustively. Rather we wished to produce a book that would be of use to people just beginning to use the technique as well as to more advanced practitioners. In this regard, we have sought to highlight techniques and applications that are of current importance, while not neglecting descriptions of approaches that may be of significance in the future. We hope that we have succeeded in this. At the same time we hope that the bibliography, with indexes classified by author and title, will make this book of value to those who may disagree with our emphasis. ACKNOWLEDGMENTS. One of us (C. G. E. ) wishes to acknowledge the en­ couragment of Professor J. A. McCloskey in undertaking this project. All four of us are grateful for the continuous and expert assistance of V. A. Edmonds in the preparation of the Bibliography. Alfred L. Y ergey Bethesda, Maryland Charles G. Edmonds Richland, Washington lvor A. S. Lewis London, England Marvin L. Vestal Houston, Texas v Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . 2. Direct Liquid Introduction Interfaces . . . . . . . . . . 5 . . . 2. 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2. 2. Operating Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2. 3. Specific DLI Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2. 3. 1. Capillary Inlets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2. 3. 2. Diaphragm Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2. 3. 3. Nebulizing Interfaces . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .

Bewitched is an odd word with which to begin a chemical textbook. Yet that is a fair description of how I reacted on first leaming of ion exchange and imagining what might be done with it. That initial fascination has not left me these many years later, and it has provided much ofthe motivation for writing this book. The perceived need for a text on the fundamentals of ion chromatography provided the rest. Many readers will have a general idea of what ion chromatography is and what it does. Briefly, for those who do not, it is an umbrella term for a variety of chromatographie methods for the rapid and sensitive analysis of mixtures of ionic species. It has become highly developed in the last decade, and while it is now routinely used for the determination of organic as weH as inorganic ions, its initial impact was greatest in the area of inorganic analysis. In the past the determination of inorganic ions, particularly anions, meant laborious, time-con­ suming, and often not very sensitive "wet chemieal" methods. In the last ten years that has changed radically as ion chromatography has supplanted these older methods.

The past decade has witnessed major advances in our understanding of the chemical composition, structure, and reactivity of the complex organic-rich fossil matter known as "coal. " Nevertheless, important scientific questions concerning molecular weight distributions, degree of crosslinking, typical duster sizes, type of interconnecting bridges, the possible role of a "mobile phase," and the nature of organic sulfur forms remain topics of heated debate. Moreover, there appears to be a notable lack of consensus regarding the overall direction and goals of structural elucidation work. Is it worthwhile to study whole coal samples, or should we separate out the various, more or less well-defined, maceral and mineral constituents before attempting to describe the structural and compositional features of coal at the molecular Ievel? Second, should there be more emphasis on key structural features and average statistical parameters, or is it necessary to identify individual chemical structures in considerable detail? From the developments of the past decade it is clear that advanced spectroscopic techniques are playing an increasingly important role in resolving difficult questions with regard to the chemical structure and composition of coal. Moreover, it has become equally clear that no single spectroscopic approach can provide all the answers but multiple techniques need to be used in a highly integrated and synergistic manner.

Experts from both academia and industry provide an overview of current research in computer-assisted analytical spectroscopy and chemometrics.

In response to the growing use of mass spectrometry in the clinical and biomedical fields, this book collects recent research involving electrospray ionization, neuronal systems, and structural modifications of proteins. The significant advances in instrumentation, methodology, experimentation presented herein will serve to expand the current concept of clinical mass spectrometry.

In response to the demands of contemporary solid material analysis-greater powers of detection, speed, depth, and precision-glow devices are receiving increased attention by specialists. This volume covers fundamental plasma processes, laser-based methods, thin film analysis, and many other processes to provide the researcher with an extensive technical reference of these devices.

- __ * ___ __ * - __ e _e __ M-A-S-S S-P-GBP-C-T-R-O-M-GBP-T-R-Y in Morse code This volume collects descriptions of selected recent developments in state-of- the-art mass spectrometric methods and reflects the broad-based approaches that mass spectroscopists apply to a variety of important clinical and bio- medical problems. One chapter reviews current mass-spectrometric instrumen- tation and techniques, and other chapters describe the use of mass-spectro- metric methods for the analysis of diacylglycerylphospholipids; modifications to DNA molecules; the characterization of variant hemoglobins; and charac- terization of urinary nucleosides. The final chapter describes the new technique of combined microdialysis/mass spectrometry. This volume represents the collected efforts of several highly productive researchers who have developed new methods and instrumentation and have applied them to current research problems, such as lipid storage diseases, cancer, hemoglobinopathies, and brain neurochemistry.The chapters in Vol- umes 1 and 2 define the outlines of clinical and biomedical mass spectrometry and attest to the flexibility and creativity of mass spectroscopists and their interaction with biologic and clinical scientists. The authors in this volume are to be congratulated for their writing efforts, their scientific vigor and rigor, their intellectual contributions, and the ex- perimental details that are described in these chapters. I thank each author for collaborating with me on the production of this volume, and I hope these chapters will help the practitioners of, and the newcomers to, the field of mass spectrometry.

The current volume covers research advances in nuclear magnetic resonance, mass spectrometry, and optical spectroscopy with emphasis on computer-assisted interpretation methodologies.

Determining the composition and properties of complex hydrocarbon mixtures in petroleum, synthetic fuels, and petrochemical products usually requires a battery of analytical techniques that detect and measure specific features of the molecules, such as boiling point, mass, nuclear magnetic resonance frequencies, etc. there have always been a need for new and improved analytical technology to better understand hydrocarbon chemistry and processes. This book provides an overview of recent advances and future challenges in modern analytical techniques that are commonly used in hydrocarbon applications. Experts in each of the areas covered have reviewed the state of the art, thus creating a book that will be useful to readers at all levels in academic, industry, and research institutions.

Determining the composition and properties of complex hydrocarbon mixtures in petroleum, synthetic fuels, and petrochemical products usually requires a battery of analytical techniques that detect and measure specific features of the molecules, such as boiling point, mass, nuclear magnetic resonance frequencies, etc. there have always been a need for new and improved analytical technology to better understand hydrocarbon chemistry and processes. This book provides an overview of recent advances and future challenges in modern analytical techniques that are commonly used in hydrocarbon applications. Experts in each of the areas covered have reviewed the state of the art, thus creating a book that will be useful to readers at all levels in academic, industry, and research institutions.

The first volume in this series is devoted to derivatization techniques in chromatography, for very obvious reasons. In gas chromatography (GC) chemical derivatization as an aid to expand the usefulness of the technique has been known for more than a decade and has become an established approach. The first chapter deals to a great extent with derivatization for the purpose of making compounds amenable to Gc. Although the discussion concentrates on pesticides, some generally valid conclusions can be drawn from this chapter. Chemistry will not be limited to the separation-it can also have a pronounced impact on the sample cleanup, another topic cov­ ered in Chapter 1. Since the introduction of coupled GC-mass spectroscopy (GC-MS), a very powerful tool, derivatization techniques have taken still another di­ rection-taking into consideration chromatographic as well as mass spec­ trometric improvement of the compounds of interest. Cyclic boronates are discussed as derivatization reagents for this purpose in the second chapter.

This textbook is an outgrowth of the author's experience in teaching a course, primarily to graduate students in chemistry, that included the subject matter presented in this book. The increasing use and importance of atomic spectroscopy as an analytical tool are quite evident to anyone involved in elemental analysis. A number of books are available that may be considered treatises in the various fields that use atomic spectra for analytical purposes. These include areas such as arc-spark emission spectroscopy, flame emission spectroscopy, and atomic absorption spectroscopy. Other books are available that can be catalogued as "methods" books. Most of these books serve well the purpose for which they were written but are not well adapted to serve as basic textbooks in their fields. This book is intended to fill the aforementioned gap and to present the basic principles and instrumentation involved in analytical atomic spectro­ scopy. To meet this objective, the book includes an elementary treatment of the origin of atomic spectra, the instrumentation and accessory equipment used in atomic spectroscopy, and the principles involved in arc-spark emission, flame emission, atomic absorption, and atomic fluorescence. The chapters in the book that deal with the methods of atomic spectro­ scopy discuss such things as the basic principles involved in the method, the instrumentation requirements, variations of instrumentation, advantages and disadvantages of the method, problems of interferences, detection limits, the collection and processing of the data, and possible applications.

Since the appearance of the first two volumes of Modern Fluorescence Spectroscopy in 1976, important advances continue to be made in both the techniques and applications of molecular luminescence. In terms of "hardware," it is only recently that the application of laser excitation to molecular fluorometry has become feasible under conditions that are analy­ tically realistic. The improvements that can be effected in sensitivity, analy­ tical selectivity, and ability to handle "difficult" samples by laser fluorometry have only begun to be exploited. Likewise, time-resolved fluorometry has received widespread use in fundamental studies (a sizable number of which deal with biological systems), but has as of yet received relatively little analytical utilization. The use of electronic array detectors offers the promise of obtaining luminescence spectra more rapidly, and perhaps ultimately with greater sensitivity, than is possible by the use of scanning instruments equipped with conventional detectors. The increasing capabilities of microcomputers and the increasing sophistication of "smart" spectroscopic instrumentation signify that much more efficient acquisition and use can now be achieved of the information contained in the "excitation-emission matrix" inherent in the luminescence phenomenon.

The Second Hidden Peak Symposium on Computer-Enhanced Analytical Spectroscopy, held in June, 1988, at the Snowbird Resort (Salt Lake City, Utah), centered around twelve keynote lectures delivered by some of the foremost experts and pioneers in this rapidly expanding field. The editor is highly indebted to each of these colleagues for contributing a chapter to the second volume of Computer-Enhanced Analytical Spec­ troscopy. The primary objective of this volume is to present a repre­ sentative cross-section of current activities in the field while balancing out the lighter coverage of some topics and areas in Volume 1. An exciting new topic, remote IR sensing, is covered in Chapters 4 and 5. Deconvolution and signal-processing methods have now been extended to UV/VIS (Chapter 1) and GC/MS (Chapter 3) applications. Furthermore, the development and testing of novel factor analysis techniques in the areas of UV /VIS and IR spectroscopy are discussed in Chapters 2 and 12, respectively. Fundamental aspects of library search techniques are presented in Chapters 7 (MS) and 9 (NMR). Chapters 6, 10, and 11 cover selected uses of expert systems in NMR, IR, and MS, respectively. Finally, an integrated expert system approach to the interpretation of GC/IR/MS data is outlined in Chapter 8. In an attempt to facilitate access to the various topics for the newcomer to the field, the twelve chapters have been organized into two main parts: Unsupervised Methods: Spectral Enhancement, Deconvolu­ tion, and Data Reduction, and Supervised Methods: Expert Systems, Modeling, and Quantitation.

The application of the Fourier transform is being seen to an increasing extent in all branches of chemistry, but it is in the area of chemical analysis that the greatest activity is taking place. Fourier transform infrared and nuclear magnetic resonance spectrometry are already routine methods for obtaining high-sensitivity IR and NMR spectra. Analogous methods are now being developed for mass spectrometry (Fourier transform ion cyclo­ tron resonance spectrometry) and microwave spectroscopy, and Fourier transform techniques have been successfully applied in several areas of electrochemistry. In addition the fast Fourier transform algorithm has been used for smoothing, interpolation, and more efficient storage of data, and has been studied as a potential method for more efficient identification of samples using pattern recognition techniques. Linear transforms have also been shown to be useful in analytical chemistry. Probably the most important of these is the Hadamard transform, which has been applied in alternative methods for obtaining IR and NMR data at high sensitivity. Even though measurements involving this algorithm will probably not be applied as universally as their Fourier transform ana­ logs, in the area of pattern recognition application of the Hadamard trans­ form will in all probability prove more important than application of the Fourier transform.

The principles of fluorescence spectroscopy are by now well established, and, after a rather lengthy gestation period, the technique is now routinely applied to a broad spectrum of problems, ranging from mechanistic photo­ chemistry to chemical analyses in biomedical and environmental systems to probes of structure and function in biological macromolecules. Phosphor­ escence spectrometry and chemiluminescence are also well-known tech­ niques; they are somewhat less well established than fluorescence (at least in analytical chemistry), but they too are receiving greatly increased appli­ cation to both laboratory and "real" problems. This is not to imply that luminescence spectroscopy, viewed in its broadest sense, is a static field. In fact, recent advances in instrumentation make it feasible to apply fluorescence to problem areas in which its use five years ago would have been unthinkable. Advances in hardware generate advances in application, and very significant progress is being recorded in the application of fluorescence (and its close relatives, phosphorescence and chemiluminescence) in the biochemical, biomedical, and environmental spheres.

From the first appearance of the classic The Spectrum Analysis in 1885 to the present the field of emission spectroscopy has been evolving and changing. Over the last 20 to 30 years in particular there has been an explosion of new ideas and developments. Of late, the aura of glamour has supposedly been transferred to other techniques, but, nevertheless, it is estimated that 75% or more of the analyses done by the metal industry are accomplished by emission spectroscopy. Further, the excellent sensitivity of plasma sources has created a demand for this technique in such divergent areas as direct trace element analyses in polluted waters. Developments in the replication process and advances in the art of pro­ ducing ruled and holographic gratings as well as improvements in the materials from which these gratings are made have made excellent gratings available at reasonable prices. This availability and the development of plane grating mounts have contributed to the increasing popularity of grating spectrometers as com­ pared with the large prism spectrograph and concave grating mounts. Other areas of progress include new and improved methods for excitation, the use of controlled atmospheres and the extension of spectrometry into the vacuum region, the widespread application of the techniques for analysis of nonmetals in metals, the increasing use of polychrometers with concave or echelle gratings and improved readout systems for better reading of spectrographic plates and more efficient data handling.

We continue in this second volume the plan evident in the first; i.e., of presenting a number of well-rounded up-to-date reviews of important developments in the exciting field of ion-selective electrodes in analytical chemistry. In this volume, in addition to the exciting applications of ISE'S to biochemistry systems represented by the description of enzyme electrodes, there is featured the most recent development in ISE'S, namely, the joining of the electrochemical and solid state expertise, resulting in CHEMFETS. The scholarly survey of the current status of ISE'S will undoubtedly be welcomed by all workers in the field. Tucson, Arizona Henry Freiser vii Contents Chapter 1 Potentiometric Enzyme Methods Robert K. Kobos 1. Introduction . . . . . . 1 2. Soluble Enzyme Systems . . . 5 2.1. Substrate Determinations 5 2.2. Enzyme Determinations . 13 2.3. Inhibitor Determinations. 18 3. Immobilized Enzyme Systems . 19 3.1. Methods of Immobilization. 19 3.2. Characteristics of Immobilized Enzymes 23 3.3. Analytical Applications with Ion-Selective Electrodes 23 4. Enzyme Electrodes 31 4.1. Urea Electrodes 35 4.2. Amygdalin Electrodes 39 4.3. Glucose Electrodes . 40 4.4. Penicillin Electrodes 40 4.5. Amino Acid Electrodes 41 4.6. Nucleotide Electrodes 46 4.7. Uric Acid Electrode 47 4.8. Creatinine Electrode 48 48 4.9. Acetylcholine Electrodes. 4.10. D-Gluconate Electrode 49 4.11. Lactate Electrode 49 4.12. Inhibitor Determination 50 4.13. Substrate Electrodes 50 4.14. Current Trends . . . .

June 1986 brought together some of the world's leaders in computer­ enhanced analytical spectroscopy at Snowbird, Utah, for what the attendees decided to call "The First Hidden Peak Symposium." With the remarkable advances in both computer hardware and software, it is interesting to observe that, while many computational aspects of spectroscopic analysis have become routine, some of the more fundamental problems remain unsolved. The group that assembled included many of those who started trying to interpret chemical spectroscopy when computers were ponderous, slow, and not very accessible, as well as newcomers who never knew the day that spectrometers were delivered without attached computers. The synergism was excellent. Many new ideas, as well as this volume, resulted from interactions among the participants. The conclusion was that progress would be made on more fundamen­ tal problems now that hardware, software, and mathematics were coming together on a more sophisticated level. The feeling was that the level of sophistication is now adequate and that it is only a matter of time before automated spectral interpretation surpasses all but the most advanced human experts.

In recent years the effort devoted to assuring both the safety and reliability of commercial nuclear fission power reactors has markedly increased. The incentives for performing this work are large since the resulting im­ provement in plant productivity translates into lower fuel costs and, more importantly, reduced reliance on imported oil. Reliability and availability of nuclear power plants, whether fission or fusion, demand that more attention be focused on the behavior of materials. Recent experiences with fission power indicate that the basic properties of materials, which categorize their reliable behavior under specified conditions, need reinforcement to assure trouble-free operation for the expected service life. The pursuit of additional information con­ tinues to demand a better understanding of some of the observed anom­ alous behavior, and of the margin of resistance of materials to unpre­ dictable service conditions. It is also apparent that, next to plasma heating and confinement, materials selection represents the most serious chal­ lenge to the introduction of fusion power. The recognition of the importance of materials performance to nu­ clear plant performance has sustained a multimillion dollar worldwide research and development effort that has yielded significant results, both in quantification of the performance limits of materials in current use and the development and qualification of new materials. Most of this infor­ mation appears in the open literature in the form of research reports, journal articles, and conference proceedings.

In 1970 when I first seriously contemplated writing a book on electron spectroscopy, I recognized the impossibility of completely reaching my desired goals. First, the field was expanding (and still is) at such a rate that a definitive statement of the subject is not possible. The act of following the literature comprehensively and summarizing its essential content proved to be a diver­ gent series. On the other hand, the field has increased to such a size that violent changes in its basic makeup no longer occur with the frequency that was present in its early days. Furthermore, the excitement of electron spectro­ scopy lies in its many-faceted interrelationships. In the era of specialization, electron spectroscopy is an open-ended subject continually bringing together new aspects of science. I wished to discuss not just one type of electron spectro­ scopy, but as many as would be possible. The book as it stands concentrates its attention on x-ray photoelectron spectroscopy, but also presents the basis of Auger electron spectroscopy and uv photoelectron spectroscopy, as well as mentioning many of the other branches of the field. A large, many-author volume might be an answer to some of these problems. However, though anyone person possesses only a limited amount of expertise, I have always enjoyed books by a single author since what they lack in detailed knowledge they gain in a unified viewpoint. I hope the final product, though limited in its attainment of these goals, will still be of some merit.

- __ * ___ __ * - __ e _e __ M-A-S-S S-P-GBP-C-T-R-O-M-GBP-T-R-Y in Morse code This volume collects descriptions of selected recent developments in state-of- the-art mass spectrometric methods and reflects the broad-based approaches that mass spectroscopists apply to a variety of important clinical and bio- medical problems. One chapter reviews current mass-spectrometric instrumen- tation and techniques, and other chapters describe the use of mass-spectro- metric methods for the analysis of diacylglycerylphospholipids; modifications to DNA molecules; the characterization of variant hemoglobins; and charac- terization of urinary nucleosides. The final chapter describes the new technique of combined microdialysis/mass spectrometry. This volume represents the collected efforts of several highly productive researchers who have developed new methods and instrumentation and have applied them to current research problems, such as lipid storage diseases, cancer, hemoglobinopathies, and brain neurochemistry.The chapters in Vol- umes 1 and 2 define the outlines of clinical and biomedical mass spectrometry and attest to the flexibility and creativity of mass spectroscopists and their interaction with biologic and clinical scientists. The authors in this volume are to be congratulated for their writing efforts, their scientific vigor and rigor, their intellectual contributions, and the ex- perimental details that are described in these chapters. I thank each author for collaborating with me on the production of this volume, and I hope these chapters will help the practitioners of, and the newcomers to, the field of mass spectrometry.

In response to the demands of contemporary solid material analysis-greater powers of detection, speed, depth, and precision-glow devices are receiving increased attention by specialists. This volume covers fundamental plasma processes, laser-based methods, thin film analysis, and many other processes to provide the researcher with an extensive technical reference of these devices.

Bewitched is an odd word with which to begin a chemical textbook. Yet that is a fair description of how I reacted on first leaming of ion exchange and imagining what might be done with it. That initial fascination has not left me these many years later, and it has provided much ofthe motivation for writing this book. The perceived need for a text on the fundamentals of ion chromatography provided the rest. Many readers will have a general idea of what ion chromatography is and what it does. Briefly, for those who do not, it is an umbrella term for a variety of chromatographie methods for the rapid and sensitive analysis of mixtures of ionic species. It has become highly developed in the last decade, and while it is now routinely used for the determination of organic as weH as inorganic ions, its initial impact was greatest in the area of inorganic analysis. In the past the determination of inorganic ions, particularly anions, meant laborious, time-con­ suming, and often not very sensitive "wet chemieal" methods. In the last ten years that has changed radically as ion chromatography has supplanted these older methods.

This book is intended both to be an introduction to techniques and applications of liquid chromatography/mass spectrometry and to serve as a reference for future workers. When we undertook its writing, we chose not to cover the field, particularly applications, exhaustively. Rather we wished to produce a book that would be of use to people just beginning to use the technique as well as to more advanced practitioners. In this regard, we have sought to highlight techniques and applications that are of current importance, while not neglecting descriptions of approaches that may be of significance in the future. We hope that we have succeeded in this. At the same time we hope that the bibliography, with indexes classified by author and title, will make this book of value to those who may disagree with our emphasis. ACKNOWLEDGMENTS. One of us (C. G. E. ) wishes to acknowledge the en­ couragment of Professor J. A. McCloskey in undertaking this project. All four of us are grateful for the continuous and expert assistance of V. A. Edmonds in the preparation of the Bibliography. Alfred L. Y ergey Bethesda, Maryland Charles G. Edmonds Richland, Washington lvor A. S. Lewis London, England Marvin L. Vestal Houston, Texas v Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . 2. Direct Liquid Introduction Interfaces . . . . . . . . . . 5 . . . 2. 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2. 2. Operating Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2. 3. Specific DLI Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2. 3. 1. Capillary Inlets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2. 3. 2. Diaphragm Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2. 3. 3. Nebulizing Interfaces . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .

The past decade has witnessed major advances in our understanding of the chemical composition, structure, and reactivity of the complex organic-rich fossil matter known as "coal. " Nevertheless, important scientific questions concerning molecular weight distributions, degree of crosslinking, typical duster sizes, type of interconnecting bridges, the possible role of a "mobile phase," and the nature of organic sulfur forms remain topics of heated debate. Moreover, there appears to be a notable lack of consensus regarding the overall direction and goals of structural elucidation work. Is it worthwhile to study whole coal samples, or should we separate out the various, more or less well-defined, maceral and mineral constituents before attempting to describe the structural and compositional features of coal at the molecular Ievel? Second, should there be more emphasis on key structural features and average statistical parameters, or is it necessary to identify individual chemical structures in considerable detail? From the developments of the past decade it is clear that advanced spectroscopic techniques are playing an increasingly important role in resolving difficult questions with regard to the chemical structure and composition of coal. Moreover, it has become equally clear that no single spectroscopic approach can provide all the answers but multiple techniques need to be used in a highly integrated and synergistic manner.