Barry L. Karger Barnett Institute Northeastern University Boston, MA 02115
Opportunities for Analytical Chemistry and Separation Science in the Biological Sciences
This REPORT discusses the past, present, and future of the relationship between analytical chemistry and the biological sciences. It is based on a portion of Barry Karger's 1990 Fisher Award Address at the ACS national meeting in Boston. In addition to the overview of the role of analytical chemistry in the biological sciences, the opportunities and directions of separation science that will take us into the next century are considered. The past It is now 25 years since one of the great analytical chemists of the twentieth century, Charles N. Reilley, received the Fisher Award. A few months after receiving his award, he summarized for this J O U R N A L his views on the state of analytical chemistry ( i ) . Analytical chemistry was a field in transition, and this was reflected in Charlie's comments. It is remarkable to read that address today and to realize that many of the things he said are still true, although they must be placed in a different context. 0003-2700/91/0363-385A/$02.50/0 © 1991 American Chemical Society
If we recall, analytical chemistry emerged from World War II in a relatively strong position. Many scientists from the Manhattan Project joined distinguished universities to set the direction of research. For analytical chemists, it was an era of inorganic and radiochemistry, with methodologies focused in these areas. Indeed, in a typical chemistry department analytical and inorganic chemistry were often combined. Recall that in the mid-1960s there was also a joint section of inorganic and analytical chemistry at the
REPORT National Science Foundation. As so rightly noted by Charlie, "Perhaps the only constant feature of science is that of change." This was never truer than in the 1960s. The era of physical organic chemistry and organic synthesis was beginning. There was an acute need to develop new analytical tools to purify and characterize complex mixtures of organic substances. As a response, NMR was beginning to emerge as a powerful tool of structural analysis; MS was being recast as an analytical technique to determine structures; GC was coming into its own as a
superb separation method for volatile species; and the first stirrings of column LC as a rapid, efficient technique were taking place. A transition was clearly in progress. Charlie's call was for analytical chemists to respond to the times. But what is analytical chemistry? An interesting question at the time (as it is today) was the definition of analytical chemistry. Charlie's definition became famous: "Analytical chemistry is what analytical chemists do." I believe he meant that analytical chemistry is defined largely by the measurement problems that workers face, and that because needs continually change, analytical chemistry (as all of science) must undergo continual change and renewal. Looking back over the past 25 years, it is clear that the analytical chemistry community did respond to the needs of the times. This past quarter-century has been one of great success, and the technological advances in analysis have been enormous. Today, a multibilliondollar chemical instrumentation industry has developed, focusing on organic analysis but including inorganic analysis as well. Chemical and pharmaceutical companies as well as those in related fields proudly support cutting-edge analytical science in their laboratories.
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REPORT A number of well-known academic centers of analytical chemistry exist, and the reputation and impact of their work are profound. The present If we turn to the present, we are again living through an intense transition period. The biological sciences especially are moving from a macroscopic descriptive field to one that examines phenomena on a molecular level. Clearly, chemistry has become a critical discipline in these sciences. The discoveries are truly breathtaking and of major significance to our lifestyles, health, and environment. It may well be that historians will look back on this era as a golden age of science. Many advances in the biological sciences are method driven. We need only look at such developments as polymerase chain reaction technology, multidimensional NMR, DNA sequencing, and high-resolution X-ray structural analysis as examples of methods development that have had a great impact on this field. The future is rich with additional opportunities. As the Nobel laureate Arthur Romberg said in an article on the two cultures of chemistry and biology, "Molecular biology appears to have broken into the back of cellular chemistry, but for lack of chemical tools and training, it is still fumbling to unlock the major vaults" (2). A multibillion-dollar industry based on modern biology has emerged. The creation of large quantities of natural or modified proteins, such as hormones, enzymes, and immunosuppressants, by recombinant DNA-derived technology has already had a great impact on our health. Such technology will also influence our food supply as well as environmental and energy needs in the future. Diagnostics that allow early detection of viral and bacterial diseases or screening of carriers of hereditary diseases is a field that is also undergoing major growth. It is clear that the basic biological research of today will lead to the industrial products of tomorrow. Many of the products derived from modern biology are or will be regulated by the federal government (e.g., by agencies such as the Food and Drug Administration or the Environmental Protection Agency). A company must obtain government approval before marketing its products. That approval includes demonstration of full analytical control of the manufacturing process, formulation, and shelf life. Indeed, it is fair to say that purification and quality control by validated analysis of recombinant products is a major expense in bringing a product to market.
At the same time, the substances to be analyzed are complex; and, more importantly, the impurities and degradation products may differ from the main product in only subtle ways. For example, in the case of human growth hormone, an important recombinant product, norleucine, may be substituted for leucine; deamidation of asparagine with backbone rearrangement or alternatively a proteolytic clip (cleavage) of a peptide bond may also occur (3). Each of these degraded species could be immunogenic, and analysis at the 0.1% impurity level may be necessary. A N A L Y T I C A L C H E M I S T R Y appreci-
ates these trends. Many of the A-page
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articles describe the use of instrumentation to solve biological problems. Also significant have been the recent introduction of Perspectives in analytical biotechnology as a part of the technical (research) section of the J O U R N A L and the appointment of an
associate editor in analytical biotechnology. This issue contains a special FOCUS section on the Human Genome Project, an I N S T R U M E N T A T I O N on bio-
sensors, and a Perspective on pulsed field gel electrophoresis. The future This discussion demonstrates an area of significant opportunity for analytical chemists: the biological sciences.
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However, at present there is a shortage of individuals who have the necessary backgrounds in both analytical chemistry and the biological sciences. This shortage will only grow in the years ahead as the pharmaceutical, biotechnology, and related industries expand. We are already seeing these trends in Chemical & Engineering News classified ads for analytical chemists, where a background in biochemistry is often accepted as a substitute. Such individuals are then retrained on the job. I would argue, however, that analytical chemists bring a critical perspective to the development of methods and that their role often can be crucial. We are responsible for seeing that the measurements are performed accurately and in a reproducible manner and that the results are convincing from a regulatory point of view. It is thus critical that there be well-trained individuals who have an appreciation for the fundamental basis of chemical measurements, including the instrumentation and the treatment of the data that are generated. These areas, along with fundamental physicochemical measurements, have been and continue to be the foundation of analytical chemistry. However, there may well be a need for greater focus in the years ahead on the application of measurements to the biological sciences. Another significant aspect of analytical chemistry—an aspect that historically has been important, and never more so than today—is the chemical and biological manipulation of synthesis to provide materials relevant to the study of biological systems. Chemical synthesis has always played a central role in analytical chemistry. In the case of separations, for example, the development of specific surfaces and complexing agents used in separation methods has had and continues to have a great impact on the field. In addition, the need to understand the context in which an analysis takes place has always been crucial. What is significant and what is trivial must be fully appreciated. The need to understand the properties of the molecules that are to be separated is of major importance. When we are dealing with biological substances, and in particular biopolymers, we need to recognize that the complexity of the molecules makes it difficult to generalize. By this time, the training of an analytical chemist would seem to be a hopeless task. A chemist needs to know instrumentation and data manipulation on the one hand, and organic chemistry on the other; and, finally, some biochemistry, molecular biology,
REPORT and biophysics need to be thrown in. What often happens today is that the first two subjects are covered, but the third receives far less attention. Yet, if we can agree that the need for analytical chemists to work in the bio logical sciences is at present great and that as a consequence of future growth more and more individuals will be hired in this area, then we must address the issue of training. At the same time, fields move rapidly, and it is quite easy for an individual to be even somewhat out of date soon after leaving graduate school. This situation calls out for a core curriculum in analytical chemis try, one that builds a foundation on which a person can respond to the chal lenges of changing needs in future years. At many universities, a student ma jors in analytical chemistry and minors in two other areas—often physical and organic or inorganic chemistry. One possible route is for an individual to minor in biochemistry. I believe that it is essential for a student to have at least a one-year graduate course in biochem istry, including a significant compo nent of molecular biology (more would be desirable). This course of study should have a strong practical compo nent. It is essential that analytical chemists have the ability to communi cate well with those in the biological sciences. A further suggestion is that the re cent Ph.D. analytical chemist consider a postdoctoral fellowship in a biology or molecular biology laboratory. Many leading molecular biologists and biophysicists have great appreciation for instrumentation and analysis. The Hu man Genome Project represents a good example of the combination of modern biology with instrumentation and ana lytical chemistry. The goal of the pro ject is to map and sequence the human genome; however, to achieve this, it is necessary to develop new technologies. As has been amply noted at various meetings—most recently at the ACS meeting in Washington last August— the opportunities for analytical chem ists in this area are great. The importance of the Human Ge nome Project to analytical chemistry goes beyond the present. If we extrapo late to the twenty-first century (when the project is to be completed), DNA analysis in all its presently known and future methodologies will be immense ly important. Major uses of gene ma nipulation, of which we only see the earliest glimmer, will emerge. One need only think of gene therapy and diag nostics for genetic diseases to recognize that analytical chemistry will be the required ingredient for proper control
of measurements. A student majoring in analytical chemistry can thus be well advised to become knowledgeable in molecular genetics, because this focus would clearly lead to a professionally rewarding career. Separation science I will now turn to some of the opportu nities and directions of separation sci ence that appear to be on the horizon for the 1990s and the twenty-first cen tury. As for analytical chemistry as a whole, many of these opportunities arise from advances in the biological sciences. By way of illustrating the trends, let
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us consider the field of electrophoresis. In the era of separations after World War II, electrophoresis and chromatog raphy were thought to be closely relat ed methods, both based on differential migration but separating on the basis of different principles. Indeed, in those days many separation scientists worked concurrently in the fields of chromatography and electrophoresis. The Nobel laureate Arne Tiselius is a notable example of an individual who conducted research in both fields. With the development of GC and the push for organic analysis, analytical separation scientists split off from bio logical scientists. Analytical chemists
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focused their attention on the field of small-molecule chromatography. From GC arose high-performance liquid chromatography (HPLC), which today is the premier separation technique for organic mixtures. While HPLC devel oped, biological scientists continued chromatography on low-pressure sta ble media, introducing the field of af finity chromatography as an exploita tion of biological recognition. Column efficiency and performance were less important in this area because the se lectivity was so high that theoretical plates were of minor concern. Because it was based primarily on specific bind ing (from a biological point of view), affinity chromatography became a tool used in the biological sciences; the ana lytical chemist hardly knew about this method as it was developed. Electrophoresis in the 1970s contin ued to be rapidly developed for biologi cal uses, and anticonvective media such as polyacrylamide and agarose gels were introduced. Most separations were carried out on a flat plate rather than by using a column because this was simple and not costly. Gel electro phoresis, although a critical tool for protein and DNA analysis and purifi cation, was not in the analytical chem istry domain. When offered at academic analytical chemistry departments, the teaching of separation methods was focused main ly on chromatography and the instru mental aspects of chromatography. Af finity chromatography, electrophore sis, and other biological separation procedures, if mentioned, were often afterthoughts. In the 1990s, continuing a trend of the 1980s, separation science in the or ganic and biological fields has merged. Capillary electrophoresis (CE), the in strumental approach to electrophore sis, is an excellent example of this trend. At the same time, affinity chro matography has become part of the lex icon of analytical systems. Thus, to be a state-of-the-art separation scientist in the 1990s, it is important to have knowledge of protein and DNA separa tions by chromatography and electro phoresis. Looking at the trends in another way, we can note that there are two aspects of separations. One is the in strumental component, involving col umn or plate operation, forced flow (pressure, electrical), detection, data manipulation, and automation. Includ ed in this area are the high-perform ance aspects of operation to achieve separations rapidly. Such issues as par ticle size, shape, and the quality of the surface necessary to achieve rapid desorption kinetics are addressed.
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REPORT In terms of efficiency, we are entering a new era of 10B-107 theoretical plates per meter (4). These efficiencies, however, are being obtained for large molecules with molecular weights of 10 5 -10 6 or greater. These developments will result in the successful resolution of complex mixtures in which separation factors of 1.002 or less are all that are necessary for baseline resolution. The second aspect is selectivity. This topic has always played a central role when dealing with closely related species. One need only think of chiral separations where organic chemists have made significant contributions to the development of separation principles for chiral species. Interestingly, this field has become even more important today as regulatory agencies consider the issue of chiral purity along with mass purity of substances. Selectivity obviously also plays a critical role when dealing with biological systems, but one difficulty in such an area is the complexity of the molecules and the fact that many different phenomena can occur simultaneously. However, affinity chromatography, as already noted, illustrates that it is possible to develop biorecognition principles of a general nature. For analytical chemists to appreciate the power of selectivity in biological systems, it is essential to have a strong appreciation of the properties of macromolecules and to understand such phenomena as protein denaturation and DNA structural motifs. Because of the diversity of problems and the complexity of the mixtures to be analyzed, two-dimensional separations will become even more important in the years ahead. An excellent example of two-dimensional separations is chromatography (or electrophoresis) coupled to MS. In this regard, we consider MS to be a powerful separation tool as well as a detector. MS has developed rapidly in the past 20 years, and its growth is unbelievably fast at the present time. Its application to biological systems—particularly peptides, proteins, and glycoproteins—is clear. The advent of high-resolution MS offers a powerful approach to separation. The coupling of other orthogonal separation procedures with each other is also very important. Such methods as two-dimensional slab gel electrophoresis—a procedure with such a high peak capacity that it is often used as a criterion of purity—have been and continue to be developed. Necessarily, issues of reproducibility and control come into play. At the same time, the coupling of two-column separation methods in which the second method is operated under high-speed conditions
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also offers a potentially powerful twodimensional approach for separations (5). In the years ahead, there is no question that the coupling of column chromatography and column electrophoresis in an automated fashion will be utilized. We need to add a word concerning detection. Obviously, one cannot determine what has been separated without the capability to determine what is present. Universal detection has its place, but all species are then observed in the chromatogram or the electropherogram. Selective detection often offers higher sensitivity and simpler separation. Here the selectivity of the detector is coupled to the selectivity of the separation system. Advances in detection methods are occurring at an extremely rapid pace, especially in the area of laser-based systems. Today, mass detection in the attomole (10~18 mol) range based on laser-induced fluorescence is routine for on-column detection. Zeptomole (10~21) and lower ranges are also being reported (6). Such levels permit separation and analysis of extremely small portions of sample, such as tissue sections. The demand for proper analytical control with this power will be extremely challenging. For example, what is a representative sample? The development of laser-based detection represents a good illustration of the trends in analysis. Often, instrumental methods (in this case, separation and detection) advance, and sample manipulation procedures are challenged to keep step. This points out the critical role of sample preparation on which the success of an analysis often hinges. As we move into the 1990s and the twenty-first century, automation will also become more critical. Automation carries with it not only computer control but also chemistries and systems that are fail-safe, all of which provide significant demands and challenges for the analytical chemist. Nevertheless, the opportunities are great. One can consider the areas of immunoassay and DNA diagnostics as fields in which automation and throughput will be significant. Conclusion We live in a period of transition with major advances of the biological sciences in many areas. These advances are driven by a molecular understanding of biological processes, and chemical measurements and methodologies are a critical component of this understanding. Consequently, analytical chemists have a major opportunity to participate in these emerging fields.
The biological sciences area clearly represents one of the significant direc tions for analytical chemists to pursue. Our era is much like that of 25 years ago. As noted by Charlie Reilley, inter disciplinary endeavors were important for the advancement of the science of analysis. The analytical chemist has al ways been part of a team, and this will never be more so than in the years ahead. The chemist's ability to com municate with people in other sciences, to appreciate their problems, and to translate them into relevant analytical methods will be important. The last 25 years have been a period of great success for the analytical chemistry community. We can grow on the current strength and have a major impact on biological science develop ments in the years ahead. I am optimis tic that our community will answer the challenges and opportunities so that 25 years from now people will look back with pride on what analytical chemis try has contributed to science.
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References (1) Reilley, C. N. Anal. Chem. 1966,38,35 A. (2) Romberg, A. Biochemistry 1987, 26, (3) Hanock, W. S.; Canova-Davis, E.; Chloupek, R. C; Wu, S-L; Baldonado, I. P.; Battersby, J. E.; Spellman, M. W.; Basa, L. J.; Chakel, J. A. "Therapeutic Peptides and Proteins: Assessing the New Technologies," Banbury Report 29, 1988; Cold Spring Harbor Laboratory, NY; p. 95. (4) Guttman, Α.; Cohen, A. S.; Heiger, D. N.; Karger, B. L. Anal. Chem. 1990,62, 137. (5) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 978. (6) Cheng, Y. F.; Wu, S.; Chen, D. Y.; Dovichi, N. J. Anal. Chem. 1990, 62, 496.
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The Journal of Organic Chemistry
solicits manuscripts that address topics at the interface of organic chemistry and biology.
W Barry L. Karger received a B.S. degree from the Massachusetts Institute of Technology in 1960 and a Ph.D. from Cornell University in 1963. He became the founding director of Northeastem's Barnett Institute of Chemical Analysis and Materials Science in 1973, and he is the James L. Waters professor of analytical chemistry. He was involved with the development of HPLC in the 1970s and is currently involved in the study of biochemical separations and analytical biotech nology with special emphasis on the field of CE.
hile such manuscripts should address fundamental problems in organic chemistry (structure, mechanism, synthesis), we encourage submission of manuscripts in which these problems are solved with the use of techniques not traditionally associated with organic chemistry (enzyme kinetics, enzyme isolation and purification, identification of active site residues, etc.). The Journal hopes to foster integrated publications in which the chemical aspects are not separated from the biological aspects. For manuscript format, see J. Org. Chem. 1990. 55 (1). 7A-10A. Send manuscripts to: C. H. Heathcock, Editor-in-Chief: The Journal of Organic Chemistry. Department of Chemistry: University of California; Berkeley. CA 94720
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