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Analytical Science at the Center of Chemistry and Beyond its Frontier

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Award Address

American Chemical Society Division of Analytical Chemistry Award for Distinguished Service in the Advancement of Analytical Chemistry, Sponsored by Waters Corporation

(Appendix on Natural Selection and References added September 26, 2000)

Roland F. Hirsch, Medical Sciences Division, SC-73
Office of Biological & Environmental Research
Office of Science
U.S. Department of Energy
Germantown, Maryland 20874-1290 U.S.A.

Introduction

In accepting this award I would first like to give thanks to my family, my teachers, and my colleagues through the years for making it possible for me to develop my abilities and for encouraging me to participate in my profession. I would not be here today if it were not for them. I am very happy to have so many members of my family here today. Tomorrow I plan to acknowledge some of my teachers and colleagues, but there are four people I must mention today: Henry Blount, Ari Patrinos, Bob Marianelli, and Ted Williams.

I must offer special thanks to the Waters Corporation. Dr. James Waters and his company not only have been noteworthy innovators in scientific instrumentation, but also have returned much to the profession and its practitioners. I am pleased that Dr. Dorothy Phillips of Waters is here today, as she has been very supportive of the science of analytical chemistry and in particular of the Division.

The first part of my talk will be about some of the changes in analytical chemistry I have seen during my years in this field. Then I wish to discuss how analytical chemistry relates to other disciplines and explain why I think our field is critical to progress in many other areas, focusing on its impact on the biological sciences. Finally, I hope to convince you-if indeed you need any convincing-that the future of analytical science is going to be even more remarkable than its past and present.

Development of Analytical Chemistry over 35 Years

My thirty-five years of professional life and service are a short span compared to the age of our field, yet I believe the changes during this time have been more dramatic than in any other comparable period.

The technological development of analytical chemistry has certainly been noteworthy, and I could spend the entire time available just discussing how, for example, lasers and integrated circuits-quite new in 1965-have not only resulted in new instruments and techniques, but have also unleashed a remarkable wave of creativity in analytical research. However, instead of cataloging these new devices and how they are used, I want to focus on the new capabilities that they have enabled.

One great advance is that it is possible to detect single atoms and single molecules in a considerable variety of samples. In 1965 there was evidence for detection of atoms using specialized microscopes or radioactivity-based measurements, but the techniques were not general. Research at Oak Ridge in the 1970s resulted in reliable detection of single atoms of many elements, and research at Los Alamos and other laboratories in the 1980s resulted in techniques for single molecule detection that are being applied to an expanding variety of chemical species . This progress has added zeptomole and yoctomole to our everyday vocabulary, words that we did not even know in 1965.

This has brought analytical chemists into uncharted waters. As pointed out by Tomas Hirschfeld in his landmark 1976 Analytical Chemistry article “Limits of Analysis” , the ability to detect very small numbers of atoms or molecules results in remarkable risks of contamination: for example, just one bacterium per milliliter is a concentration of one part per trillion by weight. Also, as the analyte concentration decreases the number of particles seen by the analytical probe becomes so small that their motion in and out of the probe volume affects analytical accuracy.

Further, we risk wrongly assuming that the particles are uniform in composition. For example, chemical analysis of a macroscopic homogenized blood sample can achieve very high accuracy and precision, but information about the chemistry of the individual cells is lost in the process. Studying the composition of cells one by one allows detection of a very small population of abnormal cells that could be an early indication of disease-but only if enough cells are studied to be sure of finding the possibly one in a hundred abnormal ones.

Great improvement has been achieved in the resolution or selectivity of analytical techniques. While procedures were available in 1965 for identifying many large molecules, doing so in the presence of very similar species was slow and expensive. Studies of environmental contamination were complicated by the inability to distinguish among, for example, the many chlorinated dioxins or between DDT and the PCBs. Today analytical techniques exist that can resolve complex mixtures with high reliability and speed, provided techniques are selected that are appropriate for the analytes of interest and the matrix.

In 1965, most analytical instruments were isolated units. A trend toward combining instrumental modes was just beginning, seeking to take advantage of the selectivity of one technique and the sensitivity and identifying power of another. The title of a recent research article in Analytical Chemistry shows how far we have come: ‘Reversed-phase high-performance liquid chromatography combined with on-line ultraviolet diode array, Fourier transform infrared, and proton nmr spectroscopy and time-of-flight mass spectrometry: application to a mixture of nonsteroidal anti-inflammatory drugs.’

Indeed, we have seen extraordinary improvements in the efficiency, speed, and reliability of our instrumentation. This is obvious, and yet I think we don’t give proper credit to the scientists and engineers of the instrument companies for the high level of performance of their remarkable products.

Data-Information-Knowledge-Understanding

I would now like to evaluate the place of analytical chemistry–and analytical chemists–in the wider world. I’d like to use the following concept: Experiments provide data, from which information can be obtained. A series of experiments yields sufficient information about a subject to add to our store of knowledge about it. Finally, knowledge from a variety of sources enables understanding with which to solve a complex problem.

For example, data from an nmr or x-ray diffraction experiment on a protein provide information about the three-dimensional structure of that molecule. Information about many such structures in a family provides knowledge about how the structure is affected by changes in composition. This knowledge, combined with knowledge from other fields, can lead to understanding the molecular basis of a particular disease and how to prevent or treat it.

In the early history of quantitative analytical chemistry there was a close linkage between those who accumulated experimental data and information and those who advanced our store of knowledge and understanding. Indeed, often the same person had both roles. Specialization gradually separated these functions, and by 1965 I believe many analytical researchers and practitioners had narrowed their focus to the first two elements in this progression. Some of this was due to the new and powerful instrumentation that was having an impact on every aspect of our field. We focused on learning what these instruments could do, while those outside our field felt the new instrumentation enabled them to do analytical work on their own. Indeed many of us in academe rarely talked to our colleagues in other disciplines. Also, much analytical teaching and research at that time was done on quite un-real samples and problems-something to which I plead guilty myself. The decline in regard for analytical chemistry at some prominent universities was due in part to these factors.

Today, many of the best analytical scientists collaborate with their counterparts in other disciplines. There are many examples. The semiconductor industry is increasingly dependent on analytical scientists working closely with materials scientists in assessing characteristics of materials as the drive to ever increasing circuit densities on chips continues. A trend in pharmaceutical research is to integrate analytical chemistry and drug discovery groups, as recently reported in Chemical and Engineering News.

In the environmental sciences, I sense an increasing realization that analytical techniques cannot today characterize the really difficult contamination problems. There is more and more collaboration between analytical researchers and the environmental problem holders. One of the speakers in the symposium tomorrow, Mark Gilbertson of the Department of Energy, will discuss this situation. Likewise, forensic science has increasingly close ties with analytical chemistry, as the day-long symposium tomorrow on detection of explosives highlights.

There is no field, however, with a greater interest in cooperation with analytical scientists than the life sciences. Tomorrow you will hear four informed perspectives on this relationship, from Jim Cassatt of the National Institutes of Health, Isiah Warner of Louisiana State University, Michelle Buchanan, soon to be the Director of the Chemical & Analytical Sciences Division at Oak Ridge, and Lee Makowski, who just moved from the National Science Foundation to the Argonne National Laboratory.

Today I would like to offer my own thoughts, with apologies that lack of time prevents me from discussing in depth the equally important impacts of analytical chemistry in the other fields I have mentioned.

When I joined DOE in 1991 the basic concept was that gene sequence determines protein structure determines biological function. Since then sequencing of DNA has become routine, due in large measure to advances in analytical chemistry. Likewise, biophysical techniques have increased in power, making it possible to determine the three-dimensional structures of large numbers of proteins rapidly and reliably. However, the link between gene sequence or protein structure and biological function is now perceived as much more complicated than in the diagram. Regulation of the expression of proteins is a complex subject in itself, and expressed proteins often are modified chemically to their actual, functional form. Further, proteins do not usually act in isolation, but rather in membranes or as parts of complexes or aggregates of small and large molecules.

The challenge for analytical chemistry is, I believe, to enable discovery of the actual chemistry that underlies biological functions. The constituents of these systems are numerous, most often at very low concentrations, and must be measured on a wide range of time scales from the sub-nanosecond to hours and longer. Clearly progress toward meeting this challenge will require close collaboration between the members of our profession and life scientists, and I am heartened by the genuine interest in such collaboration on the part of many of the best in our field.

Application to the Concept of Natural Selection

I now wish to consider a specific intersection of analytical chemistry and the life sciences. Darwin’s idea of formation of species through evolution was first published 140 years ago. There has of course been extensive discussion of his ideas and considerable change in how the theory is formulated has occurred. However, for some time the common, though not universal, view within science has been that the contemporary, neo-Darwinian version of the theory (also known as the ‘modern synthesis’) is well-established.

This situation has changed in the last decade. Two challenges have been enabled by advances in analytical chemistry joined with other disciplines.

The first concerns the concept of a tree of life: the sequential descent of species from an ever smaller number of ancestors until one goes back to the first living cell. This is commonly pictured using a phylogenetic diagram, starting from a common ancestor cell and dividing from that point into the three distinct and separate domains of life, bacteria, archaea, and eucarya.

Rapid, accurate sequencing of DNA has been a great accomplishment, for which analytical chemists share in the credit. While the human genome has received more of the headlines, the sequencing of other complete genomes, especially of microorganisms, is having a greater impact on basic biological science. Each of the two dozen microbial genomes completed to date show contains many potential genes that have no counter-part in any other sequenced genome (often as many as 30%). Yet there also are a significant number of genes that occur in selected organisms from more than one domain (bacteria, archaea, eucarya). An organism may have genes from several widely disparate sources, rather than having accumulated them through sequential inheritance as in the tree structure. One possible explanation is that this is due to lateral (or horizontal) gene transfers.

This means that how the “tree of life” looks depends on which gene is used to construct it. DNA sequences for a particular widely-observed gene could be the basis for a diagram showing a relationship among species, but the “tree” could look radically different if analytical data for a different gene were used. The suggestion of Doolittle, Martin, and others is that there is a “web” or “net” of life rather than a tree. Not only are there many horizontal crossings between domains, but there also is no single “common ancestor cell”. As stated by Doolittle: “If, however, different genes give different trees, and there is no fair way to suppress this disagreement, then a species (or phylum) can ‘belong’ to many genera (or kingdoms) at the same time: There really can be no universal phylogenetic tree of organisms based on such a reduction to genes.”

That this contradicts the current version of Darwin’s theory can be demonstrated by looking at the diagram on page 38 of Teaching about evolution and the nature of science, published by the National Academy of Sciences in 1998. The caption reads: “The ability to analyze individual biological molecules has added great detail to biologists’ under-standing of the tree of life. For example, molecular analyses indicate that all living things fall into three domains-the Bacteria, Archaea, and Eucarya–related by descent from a common ancestor.” This has been a fundamental point of Darwin’s theory–stated here by its strongest adherents. Yet the microbial gene sequence information indicates it clearly is wrong, which suggests to me that the Darwinian theory itself is fundamentally, perhaps fatally flawed.

A second question about the mechanism of macroevolution–formation of new species–by natural selection concerns the complexity of biochemical processes that occur in living cells. Analytical techniques are allowing study of more and more of these processes in vivo, confirming that cells live through meshing of complex processes, each requiring precise combinations of many molecules. It is now understood that the many biochemical pathways in a cell are highly interlocked. Further, molecules typically are complex, aggregated, bound to membranes and chaperoned as they move from one part of the cell to another and undergo chemical changes.

Rather few cellular processes are enabled solely by the presence of a single gene product. Indeed, in some cases several different proteins must be present simultaneously, or the process does not take place at all. Such a process is called irreducibly complex. It does not occur at all unless every essential protein is present. So gradual, step-by-step evolution of the process would not work, for none of the intermediate stages would be “selected” because none of the intermediate stages would be functional. I should add that this point is supported by fundamental principles of information theory, as well as recent research that concludes that random mutations cannot create complex, biologically-specified genetic information.

Natural selection has been considered by many to be the unifying principle of biology. But these and other flaws seriously compromise the theory. Explaining biology by trying to identify origins using the potentially hundreds of different trees of life or using the uncertain and unprovable mechanisms of change in the distant past has thus far in my opinion failed. No doubt some useful scientific information may result from such studies. However, I think that understanding function and its chemical basis offers a much more secure foundation for biology, and will be far more productive than the backward-looking Darwinian approach.

After all, it is understanding of function-and of the sources of malfunctions-that will lead to advances in medicine and the other fields that are dependent on biology for progress. Knowledge of the range of chemistry that enables a given function will be fundamental for this purpose. Analytical techniques will provide much of the essential information about the functional components and their dynamics in living systems. As I pointed out earlier, the chemical complexity of biological processes is great. It will require much innovation on the part of analytical chemists to fully characterize these processes in vivo. Sensing and imaging techniques combining far greater speed, selectivity, spatial resolution and sensitivity than currently available ones will be needed. The magnitude of this challenge makes me confident that the analytical sciences will be at the very center of the biology of the future.

Conclusion

It is indeed a great time to be an analytical chemist. As Freeman Dyson said, “New directions in science are launched by new tools much more often than by new concepts. The effect of a concept-driven revolution is to explain old things in new ways. The effect of a tool-driven revolution is to discover new things that have to be explained.”

I hope that my remarks have conveyed some of the enthusiasm I feel for the future of our field, that despite the remarkable developments of the past 35 years, there are many difficult and significant challenges for us for the future. While I have concentrated on the biological sciences, the challenges are equally substantial wherever knowledge of chemistry underlies the solution of problems. It will require much creativity and inventiveness for their solution. I hope that you will be able to hear the talks tomorrow by speakers who know first-hand about these challenges.

When I began in our profession a popular slogan was “analytical chemistry is what analytical chemists do”. I wish I were creative enough to offer a replacement for this phrase, which does not begin to convey what analytical chemistry is like today. However, my attention was called to a quotation from G.K. Chesterton, the English author and source of many insightful phrases, that seems appropriate: “We do not know enough about the unknown to know that it is unknowable.”

In closing, I wish to reiterate my thanks to all who have guided me and created opportunities for me to contribute to our profession, and to thank each of you for honoring me by your presence here today.

Appendix: Some Critical Points about the Theory of Evolution

(Added September 26, 2000)

1. The problem of definitions of basic terms such as ‘species’. ‘Reproductively isolated populations’ may have features that distinguish them from each other, but they often turn out to be able to interbreed when no longer isolated. Even the term ‘evolution’ has multiple meanings.

2. The complexity of the genome-proteome-phenotype relationship. Research in biochemistry and molecular biology is revealing increasing complexity in this relation-ship, beyond the capacity of ‘natural selection’ and Darwinian theory to deal with. For example, segments of DNA that are not part of any gene nevertheless appear to have critical functional roles in all living organisms; they are not ‘junk DNA’.

3. A major complication for the Darwinian theory is the existence of lateral (horizontal) gene transfer, which appears to be widespread, contradicting the fundamental Darwinian principle of linear descent with modification (a point discussed in the talk). The Tree of Life Project Root Page on the web states: “Also, recent evidence for ancient lateral transfer of genes indicates that a highly complex model is needed to adequately represent the phylogenetic relationships among the major lineages of life.” Gupta in his comprehensive review proposes a model that is radically different from the three-domain model, stating “The results of studies reviewed here indeed point to a very different evolutionary picture from the currently widely accepted one.”

4. The irreducible complexity of certain well-characterized biochemical systems (discussed in the talk). Examples include the bacterial flagellum, blood clotting, and intracellular transport systems.

5. The source of the complex, specified information contained in even the simplest genome. The information coded in a gene is complex: there are so many possible combinations of the four bases that the random probability of any particular one is vanishingly small. And the information is specified: only a very small fraction of all the possible combinations of the bases will actually provide the needed function of the gene. What is the origin of this complex specified information? Until recently it was thought that this problem of generating complex specified information could be solved by recourse to evolutionary or genetic algorithms. That hope is now dwindling as a result of the recently proven No Free Lunch theorems, which show that evolutionary algorithms fail on average to outperform blind search.

6. Even the earliest living cells for which evidence exists (3.8 billion or more years ago) are complex, yet the time for abiogenesis of them is extremely short due to extensive sterilizing bombardment of Earth until about 3.9 billion years ago. Further, many proposed theories of abiogenesis turn out to be impossible under the conditions of that period of time, and all proposed theories have serious shortcomings.

7. The inability of Darwinian Theory to account for the ‘Cambrian Explosion’: the appearance of most of the basic body plans (‘phyla’) over a very short period of time (most estimates range from 10 to 50 million years) about 550 million years ago and, especially, the nonappearance of phyla since then, despite several massive extinctions that would have provided opportunities for new body plans to gain a foothold against the remaining established ones.

8. The modest amount of direct evidence for macroevolution, and the fact that most examples given in support of the Darwinian theory really are examples of micro-evolution (spotted moths, insecticide and antibiotic resistance, Darwin’s finches).

9. The inability of Darwinian theory to account for the failure of a number of species, including humans, to follow the principle of population expanding to consume the available food (as stated by Malthus and considered by Darwin to be a key under-pinning of his theory) as well as its inability to accommodate numerous characteristics of human behavior.

Doubts also are increasing about the fundamental philosophical foundations of Darwinian theory.

References–Added September 26, 2000

(My apologies that space limitations force me to leave out many other appropriate references)

1. Hurst, G.S.; Payne, M.G.; Kramer, S.D.; Young, J.P. Resonance Ionization Spectroscopy and One-Atom Detection. Review of Modern Physics 1979, 51, 767.

2. Goodwin, P.M.; Ambrose, W.P.; Keller, R.A. Single-Molecule Detection in Liquids by Laser-Induced Fluorescence. Accounts of Chemical Research 1996, 29, 607-613; Miniaturization is also having a big impact on sensitivity of analytical techniques: see for example Sanders, G.H.W.; Manz, A. Chip-based Microsystems for Genomic and Proteomic Analysis. Trends in Analytical Chemistry 2000, 19, 364-378.

3. Hirschfeld, T. Limits of Analysis. Analytical Chemistry 1976, 48, 16A-31A.

4. Cannon, Jr., D.M.; Winograd, N.; Ewing, A.G. Quantitative Chemical Analysis of Single Cells. Annual Review of Biophysics and Biomolecular Structure 2000, 29, 239-63. There is also variation in the activity of individual enzyme molecules, see for exampleEdman, L.; Földes-Papp, Z.; Wennmalm, S.; Rigler, R. The Fluctuating Enzyme: A Single Molecule Approach. Chemical Physics 1999, 247, 11-22.

5. Louden, D.; Handley, A.; Taylor, S.; Lenz, E.; Miller, S.; Wilson, I.D.; Sage, A. Analytical Chemistry 2000, 72, 3922-3926.

6. Henry, C. Analyze This. Chemical & Engineering News 2000, 78(27), 41-47.

7. Roach, J.S. What’s in a Genome. Analytical Chemistry 2000, 72, 609A-611A.

8. Doolittle, W.F. Phylogenetic Classification and the Universal Tree. Science 1999, 284, 2124-2128; Doolittle, W.F. Uprooting the Tree of Life. Scientific American 2000 (February), 90-95; Martin, W. Mosaic Bacterial Chromosomes: A Challenge en route to a Tree of Genomes. Bioessays 1999, 21, 99-104.

9. Doolittle, W.F., op. cit.

10. Teaching About Evolution and the Nature of Science. Washington, D.C.: National Academy Press, 1998, p. 38.

11. Behe, M.J. Darwin’s Black Box. New York: The Free Press, 1996.

12. Dembski, W.A. The Design Inference: Eliminating Chance Through Small Probabilities. Cambridge Studies in Probability, Induction, and Decision Theory. Cambridge: The Cambridge University Press, 1998; Dembski, W.A. Intelligent Design: The Bridge Between Science & Theology. Downers Grove, Illinois, U.S.A.: InterVarsity Press, 1999; Yockey, H. Information Theory and Molecular Biology. Cambridge, England: Cambridge University Press, 1992; Yockey, H. Origin of life on earth and Shannon’s theory of communication. Computers and Chemistry 2000, 24, 105-123; Spetner, L. Not by Chance! Brooklyn, NY, U.S.A.: Judaica Press, 1998.

13. Dyson, F. Imagined Worlds. Cambridge, MA, U.S.A.: Harvard University Press, 1997.

14. Chesterton, G.K., William Blake. 1910. Quoted by Dembski, W.A., Intelligent Design, p. 60.

15. See for example Doolittle, W.F. The Nature of the Universal Ancestor and the Evolution of the Proteome. Current Opinion in Structural Biology. 2000, 10, 355-358.

16. Shapiro, J.A. Genome System Architecture and Natural Genetic Engineering in Evolution. Annals of the New York Academy of Science. 1999, 870 (May 18), 23-35.

17. Maddison, D.R.; Maddison, W.P. The Tree of Life Project Root Page. http://phylogeny.arizona.edu/tree/life.html See also reference 8, above.

18. Gupta, R.S. Protein Phyogenies and Signature Sequences: A Reappraisal of Evolutionary Relationships among Archaebacteria, Eubacteria, and Eukaryotes. Microbiology and Molecular Biology Reviews. 1998, 62, 1435-1491.

19. See reference 11.

20. See reference 12.

21. Wolpert, D.; Macready, W. No Free Lunch Theorems for Optimization IEEE Transactions on Evolutionary Computation. 1997, 1 (1), 67-82; Culberson, J. On the Futility of Blind Search: An Algorithmic View of ‘No Free Lunch’. Evolutionary Computation. 1998, 6(2), 109-127.

22. Rosing, M.T. 13C-Depleted Carbon Microparticles in >3700-Ma Sea-Floor Sedimentary Rocks from West Greenland. Science 1999, 283, 674-676; Kerr, R.A. Early Life Thrived Despite Earthly Travails. Science 1999, 284, 2111-2113.

23. There are numerous articles on this issue, of which the following are a few recent examples: Levy, M.; Miller, S.L. The Stability of the RNA Bases: Implications for the Origin of Life. Proceedings of the National Academy of Sciences U.S.A. 1998, 95, 7933-7938; Orgel, L.E., The Origin of Life-A Review of Facts and Speculations. Trends in Biological Sciences. 1998, 23, 491-495; Shapiro, R. A Replicator was not Involved in the Origin of Life. IUBMB Life. 2000, 49(3), 173-176; See also Lahav, N. Biogenesis: Theories of Life’s Origins, Oxford: Oxford University Press, 1999.

24. Kerr, R.A. Evolution’s Big Bang Gets Even More Explosive. Science 1993, 261, 1274-1275; Knoll, A.H.; Carroll, S.B. Early Animal Evolution: Emerging Views from Comparative Biology and Geology. Science, 1999, 284, 2129-2137; Adoutte, A.; Balavoine, G.; Lartillot, N.; Lespinet, O.; Prud’homme, B.; de Rosa, R. The New Animal Phylogeny: Reliability and Implications. Proceedings of the National Academy of Sciences U.S.A. 2000, 97, 4453-4456. Also, complications in the classification of organisms within phyla are arising out of DNA sequencing studies, for example: Eeckhaut, et al. Myzostomida: A Link Between Trochozoans and Flatworms? Proceedings of the Royal Society (London) B 2000, 267, 1383-1392.

25. Wells, J. Icons of Evolution: Science or Myth? Washington, D.C.: Regnery Publishing, 2000.

26. Stove, D. Against the Idols of the Age. Kimball, R., editor. New Brunswick, N.J. U.S.A.: Transaction Publishers, Rutgers-The State University, 1999; Kagan, J. Three Seductive Ideas. Cambridge, MA, U.S.A.: Harvard University Press, 1998.

27. Three scholars offering distinctive perspectives: Johnson, P.E. The Wedge of Truth. Downers Grove, Illinois, U.S.A.: InterVarsity Press, 2000; Carroll, R.L. Towards a new Evolutionary Synthesis. Trends in Ecology & Evolution. 2000, 15, 27-32; Shapiro, J.A. A Third Way. Boston Review. 1997, 22(1). http://bostonreview.mit.edu/ BR22.1/shapiro.html