chromatography, today and tomorrow

Analytica Chbica Acta, 283 (1993) 309-319 Elsevier Science Publishers B.V., Amsterdam

309

Chromatography, today and tomorrow

Georges A. Guiochon

Department of Chemistry, University of Tennessee, KnoxviUe, TN 379964600; and Division of Analytical Chemistry, Oak Ridge National Laboratory, Oak Ridge, TN (USA)

(Received 7th October 1992)

Ah&act

The history of chromatography is briefly reviewed. Current trends are documented, pointing to the advanced degree of maturation of the method. Iti future as a research area results from its unique position as an extremely powerful technique of investigation of the thermodynamics and kinetics of phase equilibria, in the same time as it is the most powerful method of separation available in the life sciences.

Keywords: Chromatography; History of chromatography

A number of reviews have been published recently under a similar title [l-4]. The basic rules of the exercise seem to call for a thorough review of the present status of the method, followed by a list of the unresolved problems as seen by the author, and a more or less careful extrapolation to the future of the trends observed over the recent past. Given the poor state of the art of crystal ball detectors, and the current lack of attention devoted to the development of divina- tion methods, it is not surprising that the quality of the latter part rarely measures up to that of the first one. The human mind experiences tremendous difficulties at imagining what could happen besides the normal continuation of the present course of events.

Most people extrapolate along the slope of the current trend. Only the most gifted and best trained in the art of forecasting can take the current curvature into account. Almost by definition, new discoveries cannot be forecasted, although their annunciation would be the best pos-

Correspondence to: G.A. Guiochon, Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600 (USA).

sible contribution of an exercise such as the one I am attempting. As a particularly striking example, we can refer to the remarkable development of the personal computers in the early 198Os, to the change they made in the very concepts of modern instrumentation, and to the costly deci- sions made by companies whose managers did not anticipate this development when the first inexpensive microprocessors appeared in the late 1970s.

After this careful disclaimer, and noting that the unique crystal-ball detector known a is currently borrowed by the Department of Eco- nomics, I will follow the traditional approach. We have to remember first that chromatography 561 is celebrating its 90th birthday this year, while gas chromatography reaches the mature age of forty [7]. During this long time span chromatography has evolved, spread and is maturing. It is useful to briefly recall the salient steps of this impressive

a This detector belonged to Napoleon I, who inadvertently dropped it on the ground under his tent the night before Waterloo. It is now conserved in the Museum of the Ecole Polytechnique (Palaiseau, France).

0003-2670/93/$06.UCl 0 1993 - Elsevier Science Publishers B.V. Ah rights reserved

310 G.A. Guiochon /Anal. China. Acta 283 (1993) 309-319

TABLE 1

Stages of development of a technique

- Discovery of a new technique - Spread of the discovery - Commercialization of the proper instruments - Maturity of the technique - Inclusion in the cultural background

career. Table 1 summarizes the various steps in the evolution of a technique [l].

DISCOVERY OF CHROMATOGRAPHY

Almost everybody knows now the sad life story of Tswett [5,6]. What is not fully realized, however, is the extreme care which was paid by Tswett to experimental details, and the considerable grasp he had of the fundamental problems encountered in the use of this new analytical method [8]. Unfortunately, published in Russian in a Polish Journal of Botany [9,101, the first papers languished a long time on dusty shelves. The PhD dissertation escaped oblivion and the destructions of a civil war because a copy was passed on to Willstitter [ll], who had a German translation made and eventually gave it to Kuhn.

Eventually, Kuhn used Tswett’s results to begin the series of investigations which lead to the rebirth of chromatography [12]. Plant pigments are extremely sensitive to isomerization, and to various degradation reactions. They do not sur- vive well to extended contacts with silica. The first chromatograms were obtained by Tswett with finely divided calcium carbonate, a very gentle adsorbent. This detail escaped the German chemists in their earlier attempts at reproducing Tswett’s results using silica gel. This explains the early hostility of Willstatter 181 towards the method, and why, rather typically, the new technique faded away after a brilliant discovery was made. The method began to take off only thirty years later, after Kuhn and Lederer had over- come the artifact problems.

Note that already at this early stage, chromatography was the realm of biochemists. A botanist interested in the composition of plant

pigments, or a chemist involved in the unraveling of the structure of carotenoids would now be called biochemists. Truly, chromatography belongs to biochemists. Some of the current trends we are witnessing now and that we will discuss later are merely the continuation of an original inclination attested by the long list of affairs chromatography had with biochemistry. The period that follows the rediscovery by Kuhn and Lederer shows a rapid succession of progress and innovation, sanctioned by a long series of Nobel prizes. The work of Martin and Synge on paper chromatography [131, followed ten years later by the development of gas chromatography [7], already seeded in the original paper of Martin and Synge, are examples of the many fields which were explored and the techniques which were developed before, during, and immediately after World War II.

THE SPREADING OF CHROMATOGRAPHIC TECH- NIQUES

The spreading of chromatography as an analytical technique has proceeded by leaps and bounds. By birth, chromatography is a 19th century technique, which could have been understood, or at least assimilated and used, by the Founding Fa- thers of chemistry. In contact with the electronic revolution of the 196Os, it undertook a remarkable evolution which transformed it completely. This is illustrated, for example, by the progressive replacement of “conventional” by “high performance” column liquid chromatography a. In the first period, the instrumentation was minimum. A glass column was packed, and the mobile phase was left percolating under the driving force of its mere weight. Fraction collection, off-line analysis, lack of control, slow analysis, and-intensive human labor were the rule. The kinetics of chromatographic phenomena was not studied. During the second period, all the modern conquest of electronic based instrumentation were progres-

a HPLC has been used to mean many different things during the last 20 years, high-pressure LC, high-performance LC, or high-price LC.

GA. Guiochon / AnaL Chim. Acta 283 (1993) 309-319 311

TABLE 2

Implementations of the chromatographic principle

- Gas-solid chromatography - Gas-liquid chromatography - Supercritical fluid chromatography - Normal phase liquid chromatography - Reversed-phase liquid chromatography - Hydrophobic interaction chromatography - Ion-exchange chromatography - Size-exclusion chromatography - Ligand-exchange chromatography - Affinity chromatography - Thin-layer chromatography

sively included in the chromatograph. In present- day chromatographs, detection is on-line, the elu- ent is pumped through the column, most parameters are controlled, computer data acquisition and handling have become universal. While the equipment has become automatic, the analysts are leaving the laboratory for offices.

Indicative of the power of the chromatographic principle, is the rapid multiplication in the number of the different techniques imple- menting it, a number which increased from a few to a dozen in the 1950s and 1960s (Table 2). Each of these techniques, in turn, underwent the same cycle of discovery, spreading, explosive growth, commercialization and maturity. Some chromatographic techniques have even undergone the cycle twice. As explained above, liquid-solid chromatography, the very technique discovered by Tswett was entirely changed and rejuvenated by the incorporation of the developments which had made gas chromatography such a successful technique in the 1960s. Size exclusion chromatography appeared a mature technique in the early 1970s. It has continued developing at a steady pace by putting to use the progress made in liquid-solid chromatography (instrumentation, data handling techniques), and especially in the design of new stationary phases.

Three types of fluids with widely different sets of physico-chemical properties, liquids, low and high density gases (those including supercritical fluids) can be used as the mobile phase. A wide

variety of retention mechanisms, including all types of molecular interaction known, have been studied, and appropriate stationary phases made available for their application. Adsorption remains the premier. retention mechanism, having spawn at least five different methods, normal and reversed-phase liquid-solid chromatography, hydrophobic interaction chromatography, besides the classical gas-solid and supercritical fluid- solid chromatography. Even the total absence of molecular interactions in the stationary phase has found its place in the array of possibilities; it is the basis of size exclusion chromatography. Again, we must observe that biochemists have been at the origin of most of these methods. All this activity has created an explosion in the number of papers published, hence the number of citations, reviews, abstracts, books and computer programs. This fact has not been lost on the publishing companies.

POPULARIZATION OF CHROMATOGRAPHY

Whichever standard we use to measure it, the number of papers published in analytical chemistry journals, whether broad-based or dedicated to chromatography, has increased most rapidly between 1950 and 1980. The current trend shows certainly the approach of a maximum (Fig. 1).

Fig. 1. Number of papers published yearly by the Journal of Chromatography since its foundation in 1958.

312 GA Guiochon /Anal Chim. Acta 283 (1993) 309-319

Quite significantly, fewer and fewer papers deal with instrumentation or with fundamental issues, although every now and then a spur of activity is still observed on some special problem. On the other hand, more and more papers discuss new applications of one of the chromatographic methods to the solution of these analytical problems which were ignored yesterday, have become critical today, and will be mere routine tomorrow.

Obviously, this type of activity is necessary in many industries, or for countless applications in academic or industrial research, and the publication of its results, being of potential use to many, is legitimate. However, method development or the investigation of technological improvements is not the main stay of academic research. So, the rising tide of application papers is probably the result of the conjunction (i) of a publication system geared to handle a massive number of papers, and needing material to process; (ii) of the large number of new method developments which must be studied each year because of the expand- ing needs of analysis in the life sciences and related industries; and of (iii) the considerable effort which is still required to develop these methods, and which justifies the writing of a paper, at least in the author’s mind. The statisti- cal studies of Giddings [14,15] and Oros and Davis 1161 on the resolution power of a column and the number of components which are likely to be resolved on this column explain the fundamental source of the difficulties of method development.

Be as it may, we can safely state that the main thrust of research in chromatography is now re- turning to biochemistry, after having for many years laid in the area of physical chemistry where it was the central purpose of the activities of many scientists from a wide variety of walks. Most fundamental chromatography problems are solved now. What are not solved, are problems in other fields of chemistry that the chromatographer has to elucidate in order to perform separations. Most of them are problems of the thermodynamics of phase equilibria, and problems of material sciences. They revolve around the key question of chromatography, how to make and use better stationary phases.

THE COMMERCIALIZATION OF CHROMATOGRA- PHY

More striking still than the explosion in the number of papers, is the blossoming of a large market, supporting a multi-billion dollar industry, and served by several dozen companies of widely different sizes, selling instruments, accessories, dedicated products, and services. A number of instrumentation giants have come into chromatography through their own research and development effort, but more often by buying successful small companies created by imaginative, persistent inventors. Many medium size companies have been started by far-sighted analytical chemists, and produce highly reputable instruments. They often survived with difficulties the loss of their founder, especially when it resulted from a sale and lead to the misguided efforts of the purchasing company at boosting revenues. A vast number of small companies make or sell the variety of instruments, stationary phases, accessories, chemicals, gadgets, tubings, valves fittings, etc. that a chromatographer needs.

A single visit to the annual Pittsburgh Confer- ence on Analytical Chemistry and Applied S’c- troscopy (which had to add Analytical Chemistry to its name because chromatographers were out- numbering spectroscopists) suffices to convince anyone of the huge size and the vitality of this industry. The visit of any trade show on instrumentation the world over confirms the reality of this conclusion. Now, all this industry is here to stay, whatever happens to the research field of chromatography. Not long ago, there were still more money spent on buying gas than liquid chromatographs, although the papers published on liquid chromatography outnumbered those on gas chromatography years earlier. But the market of chromatographic instrumentation has become a replacement market. To understand what this means, we have to remember that we still buy balances, but we rarely meet to discuss the science of weighing.

What may be in doubt, however, is the future of another aspect of the popularization and commercialization of chromatography, the symposium industry. The number of these meetings has in-

G.A. Guiochm /AnaL Chim. Acta 283 (1993) 309-319 313

creased rapidly over the years. If they have contributed usefully to spread the technique, they have also largely contributed to fragment it, or rather to consolidate its fragmentation. For example, we see rarely specialists of affinity chromatography in I-IPLC symposia. Ten years ago, I collated the lists of the attendants to I-IPLC%l (ca. 600 people) and to the symposium on affinity chromatography organized the same year in Strasbourg (ca. 300 people), There were only 2 people whose names were on both lists! Symposia on planar chromatography, open tubular column chromatography, microcolumn chromatography, supercritical fluid chromatography, chiral chromatography, preparative chromatography, have multiplied, while each and every meeting on analytical chemistry needs one or several sessions on chromatography, and meetings on technique X like a session on X-chromatography coupling. You may not think seminars are a business. My travel agent does. I wonder how long this will last, and whether the reduction in attendance observed at minor meetings this year is the fruit of the reces- sion or the harbinger of a new trend.

MATURITY AND ITS PROBLEMS

History tells us that people who live through a transition period have had always great difficulties to observe it. We, chromatographers, are in such a period for two reasons. Our very field is in transition, and the world around us is also in transition, causing analytical chemistry to spread, develop and evolve. The present crisis of our technical society, induced in part by the fatigue of its actors, in part by the immensity of the new problems it is causing to itself, creates new needs. Some of them can be solved only through more frequent or more intense uses of chromatography. So, while chromatography is maturing and slowly fading as an area of fundamental scientific research, society is requiring from us new progress, and wants many more of us. This will provide for what I believe will be an intense flurry of activity in the short or medium term, may be a dozen years, followed by a rapid melting in the background of chemical culture.

Driving force for the expansion of research in chromatography

Today, research in chromatography remains very active in several areas involving the thermodynamics and kinetics of the retention mechanisms, the fundamentals of method development (including optimization and the statistics of band overlap), the extension of chromatographic separations to new classes of compounds of biochemical importance, large scale preparative chromatography and microcolumn technologies. An increasing fraction of this effort, however, is devoted more to technological development than to fundamental problems.

The reason for the extraordinary level of interest and funding we have received in the past twenty years and are still receiving now is the constant stream of new legislations boiling out from our political centers. In the United States, the innumerable Acts watching over the safety of our foods, drugs and cosmetics, the safety of our air, water and environment, the safety of our industrial practices, have made necessary the multiplication of countless analysis types (Table 3a). Most of them require the use of chromatography, alone or in combination with other techniques. Since we have decided that we should be in perfect health and live forever, the mainte- nance of our body requires also analyses in increasingly large numbers, and analyses of an ever deeper complexity. As we, academic chromatographers and biochemists, make more chromatographic analysis possible, we feed a system which, through the combination of their potential useful- ness and the threat of malpractice liability, forces physicians to require more analysis, thus making necessary the employment of more chromatographers. Although we have lost many jobs to computers in those last ten years, we have created many more through the operations of the legal system. Because of the increasingly complex requirements of a more and more sophisticated

a Understanding what it is exactly that the laws require to do, when and how can keep busy teams of lawyers and chemists which many medium size companies can ill or even no longer afford [see A.M. Thayer, Chem. Eng. News, 38 (1992) 91.

314 G.A. Guiocbn /Anal. Chim. Acta 283 (1993) 309-319

TABLE 3

List of US acts a relevant to the production and handling of chemicals

Federal food, drug and cosmetic act Toxic substances control act (TOSCA) Clean air act Clean water act Resource conservation and recovery act (RCRA) Safe drinking water act Occupational safety and health act Hazardous materials transportation act Comprehensive environmental response, compensation

and liability act (CERCLA or Superfund) Federal hazardous substance act Federal insecticide, fungicide and rodenticide act (FIFRA) Consumer product safety act Pollution prevention act of 1990 Emergency planning and community right-to-know act

(Part of Superfund amendment and reauthorization act, or SARA) Poison prevention packaging act Ports and waterways safety act Marine protection, research and sanctuaries act

a Many of these acts have been modified by amendments passed later (e.g., SARA). This greatly enhances the complexity of the legal field.

society, new demands will continue to fuel chromatographic research for a long time. Analytical chemistry and especially chromatography is a field full of promises, because it holds one of the most important keys to safety, the goal of our modem society.

Is there any reason for this process to stop before everybody has become a chromatographer, or before we know how to detect a molecule of any thing in any sample? Obviously there is, the question is when. Before discussing it, let us review the problems we have to face and solve to accomplish our mission. I will divide these problems into those which are purely chromatographic, and those which are found in other areas of science and technology, where we have to take the answers from. These are mainly phase equilibrium thermodynamics and detection methods.

Thermodynamic problems Chromatographers would like, and will need,

to be able to call a computer data bank and

determine rapidly the retention data of some new components on a series of phases. This could be done by selecting data previously measured by another scientist on the same phase or on a similar one from some computer file, by correlat- ing data previously measured for some similar components on the same, or a similar phase, or by using some computer program based on correlations between retention data and chemical structure. This would save considerable time and avoid wasting precious chemicals in the acquisition of new data. It should be emphasized that now is the time to invest in any project whose results will contribute to reduce wastes later.

Many current difficulties arise from our lack of understanding of retention mechanisms a. This has nothing to do directly with chromatography, and is truly a thermodynamic problem. The trou- ble is that so many chromatographers have un- knowingly turned themselves into thermodynami- cists without really knowing the basics nor the tools of this field. Whether gas or liquid chromatography is used, we still cannot predict retention times, retention factors, nor separation factors. Many of these problems arise because we do not have standard compounds or mixtures, which is reasonable as the variety of our problems is just too immense, and because we do not have enough retention data, which may seem surprising but is too real. There are few collections of good retention data, and most of them are insufficient to properly characterize the stationary phases. The work of McReynolds [17] and Park et al. [18] should be continued and expanded. Chemomet- rics offers us very powerful tools which we cannot really use for the lack of the relevant data. Unfor- tunately, many chemometricians seem to be con- tent to acquire data for fiftish monofunctional components, and use that data set to train their procedure to identify a few more. We need no more chemometric studies now; we need chro-

a In a recent symposium on Fundamental Aduances in Liquid Chromatography (Washington, DC, August 19921, a group of a dozen well-known leaders in the field were unable to agree on a definition of what exactly is the stationary phase when chemically bonded silica is used as the column packing.

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matographic studies using chemometrics. The lack of sufficiently large retention data

bases prevents us from normalizing stationary phases, from drawing meaningful comparisons, or from extending the work of Rohrschneider [19]. In the same time, the number of stationary phases available increases rapidly, and it becomes impossible to perform any systematic investigation to select the best phase system for the solution of but the most complicated problems. It is not even possible to require a normalized set of retention data to characterize new phases. These conclu- sions apply to all types of chromatography, gas (GC), liquid (LC) or dense gas (SF0 chromatography, whatever their exact retention mechanism (Table 2).

In GC, the role of adsorption at the gas-liquid interface is still misunderstood and difficult to account for. The choice of standard solutes is made difficult by the rapid variation of their vapor pressure, hence retention volume, with temperature. Several series of standard solutes would be necessary, but the choice of those used at high temperatures is especially difficult. To increase their vapor pressure, we need to increase their molecular weight, but cannot simply take higher homologs: the polarity would de- crease with increasing chain length. On the other hand, polyfunctional solutes are difficult to handle in all but the most sophisticated correlations. The use of polyfunctional solutes in which the same group is repeated twice or three times and these groups are separated with a short alkyl leash appears as an attractive solution (e.g., at high temperatures, methanol is replaced by 1,3- propanediol, acetonitrile by succinonitrile, ben- zene by dibenzyl).

Two other problems deserve serious attention in GC. First, as shown by Kersten and Poole [20], there are no stationary phases available which are strong proton donors. This is most surprising in view of the several hundred stationary phases described in the literature, many of which being very similar. It might be due to the poor thermal stability of such compounds, but a systematic investigation would certainly be worthwhile. Fi- nally, it is surprising to see how little interest has been devoted to the use of computers in method

development, optimization, data handling and correlation with computers in GC, especially compared to what has been done in LC in the recent past. But may be no new methods are developed in GC any longer [l].

In normal phase liquid chromatography, we have no adsorbent of standard, reference activity. We cannot predict correctly the change in retention factors occurring when we replace the strong solvent in a binary mobile phase by another one at constant eluotropic strength, nor even the changes in separation factors. In reversed-phase LC, the dependence of the retention factor on the composition of a binary mobile phase is no better understood. We know that the linear dependence of log k’ on the composition is simplis- tic, but we have no better model yet. More sophisticated approaches, such as the unified theory of retention of Martire and Boehm [21] and the solvatochromic theory as used by Cheong and Carr [22] and Johnson et al. [23] have not yet been applied to the full of their potentiality. The inability of chromatographers to predict the variation of the separation factors with the composition of a multi-solvent mobile phase prevents them from anticipating peak cross-over during the optimization of the mobile phase composition.

Needless to say, considerable work will has to be performed before we understand retention mechanisms better. The most sensible approach would probably start by studying selective mechanisms and try to predict the relative retention of closely related compounds. This way, most non- selective molecular interactions can be ignored, as they cancel out. A case in point is the separation of enantiomers, for which all behaviors are identical, except for what concerns the enantio- selectivity. But if progress has been made recently along these lines for phases using selective com- plexation or similar interaction types, we do not have much of an inkling on what controls the selective separation mechanism with cellulose and cyclodextrin phases.

Finally, as far as supercritical fluid chromatography (SF0 is concerned, the use of dense gases as the mobile phase provides first and foremost a unique opportunity to learn more about the

316 G.A. Guiochon/AnaL Chim. Acta 283 (1993) 309-319

physico-chemical properties of those fluids, which are still poorly understood. We know that the fact that the mobile phase is supercritical or not has nothing to do with the separation mechanism. All phase equilibrium properties vary smoothly with the gas density around the critical point. What counts is the gas density, which controls molecular interactions. While we begin to understand retention in high density carbon dioxide through the work of Yonker and Smith [24], Schoenma- kers [25] and Mar-tire and Boehm [21], the physical basis of the influence of polar modifiers is still controversial.

Even simple parameters, such as the diffusion coefficients of solutes in mixtures of associated solvents like water-methanol or water-acetonitrile which is a function of their composition, are impossible to predict simply. The classical correlation equations provide often no more than the proper order of magnitude.

To conclude this section, I think that there is a great future for research in phase equilibrium thermodynamics, in connection with the retention mechanisms used in chromatographic separations. Already an important part of the true research done in chromatography belongs to this area. The investigation of mass transfer properties is less advanced, but holds at least as many promises.

Chromatographic problems There are not many unsolved problems left

which can be properly qualified as chromatographic. There remain a few nagging problems, and much mopping up to do, however. This in- volves particularly problems related with the op- eration of cohunns under non-linear conditions, whether for preparative applications or to perform particular analysis. This includes also some analytical problems, and a few other problems which are instrumental in nature.

So far, multidimensional chromatography has attracted much more interest among academics than practitioners. It is not clear whether analysts are too preoccupied by their current problems, or happy enough with the separation tools presently available. In most cases, what is called multidimensional analysis is truly mere multi-column

analysis. Compared to a real bidimensional analysis, where component zones are spread over the entire plane, they involve only separations along too perpendicular narrow bands in the plane, thus affording much less separation power. Bi- and tri-dimensional analyzers could provide either much faster analysis or much higher resolution power. The question is whether there are any analytical problems requiring such progress. It does not seem that the pressure to develop that kind of technique has been great over the last ten years, in spite of the potentiality of the method, which has been clearly illustrated by Giddings [14] and Oros and Davis [16].

Preparative chromatography is still in the early stages of development. At present, the technique seems to be in a stage intermediate between spreading and popularization. The advantages, drawbacks, and limitations of several possible implementations of the method, overloaded elution, displacement, annular rotating column, simulated moving bed, are not yet clear, nor which segment of the market they can claim for [26]. Packing and operating large diameter columns is a complex process. We can no longer assume the radial homogeneity of the column, and we have no simple way to take radial heterogeneity into account. There is little information available regarding either the degree of packing homogeneity which is really achieved, or the influence of packing homogeneity on column performance. A number of facets of the problem have been investigated. The synthesis of these results and wider scope investigations are required. This is an area where the combination of modelling, computer simulations and experiments will be particularly fruitful.

Among the analytical problems which are still under active investigation, the most challenging by far is the analysis of peptides and proteins. Because these compounds are so numerous and their physico-chemical properties vary so very widely, we cannot expect any general method. The problem is complicated by the fragility of their tertiary structure, key to their biological activity. Depending whether pure analytical information is sought, or whether the recovery of purified material for further biochemical investi-

GA. Guiochon /Anal. Chim. Acta 283 (1993) 309-319 317

gation is needed, the separation method can be aggressive or must be gentle towards this strnc- ture. Combinations of size exclusion, ion-exchange and hydrophobic interaction chro- matographies could achieve very powerful separations in short periods of time, if they could be combined into a tridimensional process. We are currently observing an intense proliferation of analytical schemes taking place in this area. The acute need for streamlining separation method- ologies for protein analysis will surface soon, and true multi-dimensional analysis is probably the most effective way to approach it.

There is a growing recognition of the existence of the many artifacts that take place during a chromatographic analysis, a fact that had been forgotten all these years when LC was essentially applied to small molecules, mostly of industrial origin, or to environmental samples. Tswett had recognized them [9,10], and they slowed the adoption of chromatography by German chemists [12]. The causes of these artifacts and the conditions under which they are most prone to take place need to be better identified. They can be extremely damaging, for example in most applications of LC in the life sciences. Ignorance and an excessive confidence in your own skills and in the performance of the procedures used magnify the consequences of these damages.

Surprisingly, there are still a few nagging problems in GC which have been left behind when research in this area became narrowly focussed on the technology of coating quartz tubes with immobilized stationary phases. One of them is the prediction of exact values of the retention times in programmed temperature GC from isothermal data. The equation relating these different parameters cannot be solved in closed form, but the solution is easily calculated [27]. Nevertheless algorithms for the calculation of these solutions are not available in chromatographic data packages. Similarly, by contrast with LC, surprisingly little has been accomplished in the area of GC optimization. The simple extension of the approaches used fruitfully in LC has not even been investigated. It seems that, because GC samples are on the average simpler, and GC open tubular columns have a much larger separa-

tion power than LC packed columns, great re- turns could be expected from such studies.

Instrumental problems As observed by Lochmiiller [l], we see a grow-

ing demand for inexpensive, high performance equipment. The science of designing and manu- facturing scientific equipments has made great progress in the last ten years. Minor adjustment of performances may permit considerable savings. This is particularly important for the instruments sold to the bioscientists who seem to be less demanding on certain types of high performance instruments, such as chromatographs with quadruple gradient programmers, and fast scan- ning UV diode-array photometers.

There has not been a real innovation in instrument design for years. Still some needs are un- met. I sometimes wonder whether there has been a better gas chromatograph than the short-lived Perkin-Elmer 226 of the mid 1960s. The weakest parts of the current chromatographs remain the sampling system in both GC and LC, and the LC detectors. The sensitivity and dynamic linear range of the UV detector have nearly reached their theoretical limits [28]. No principle has been suggested yet on which could be based the design of an LC detector having performance (i.e., detection limits and dynamic linear range) compara- ble to those of the flame ionization detector in GC. In a related area, the development of detectors based on polarimetry and circular dichroism will bring a useful complement to the present detector collection. A most attractive addition to the pride would be an instrument specially de- signed to perform multidimensional chromatography as easily as LC column chromatography. This is currently carried out.

Ten years ago, there was a raging controversy regarding the miniaturization of the chromatographic columns. Optimistic analysts kept predict- ing the coming of age of open tubular columns for LC, or at least the wide-spread use of microcolumns. Except for some attractive but yet academic works, little progress is to be seen on the instrument market. A few dedicated pumps, gradient generators and detectors are available in what is still considered as a small market niche.

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The pessimists who kept insisting on the nature and magnitude of the problems to be solved were, unfortunately, right. Slowly, however, knowledge is acquired and accumulated on the design and behavior of microcolumns and the situation will probably change within the next ten years. After all, miniaturization permits faster analysis and/or higher separation powers. It would allow considerable reductions in the amount of waste generated, thus satisfying ad- ministrative requirements. The market keeps pushing in these directions, slowly but decisively.

Condusbns Although chromatography has now become a

mature technique, it enjoys a unique cross-road position which will ensure it a bright future before it is entirely assimilated in the cultural background of chemists.

On the one hand, chromatography is an extremely powerful tool for physico-chemical studies of nearly all molecular interactions. All phase equilibria, their thermodynamics and kinetics can be studied more easily, accurately, rapidly than with any other technique. The method can be used as easily to carry out measurements at extremely low concentrations (below 1 ppb) as to make them at very high concentrations (close to 100%). It will be a long time before this gold mine is exhausted.

On the other hand, chromatography is the premier separation method for molecular sciences. As biochemistry and all the life sciences are becoming more and more molecular, they will generate a nearly endless source of challenging analytical problems. The same chromatographic principle permits the production of purified amounts of biochemicals with negligible concentrations of any specified impurities. The development of suitable procedures is also a major task of the immediate future.

More importantly, however, chromatography is one of the critical tools required for the application of the tide of rules which aim at giving us total safety and a long life. Legal requirements regarding the handling of toxic chemicals (all chemicals are presumed to be so, until proven innocuous), and the monitoring of exposure to

them by workers and by the public at large is the powerful drive towards the development of more sensitive trace analysis, highly selective detection, cheap microchromatographs able to monitor indi- vidual exposure to certain compounds, and complex instruments able to separate and quantitize hundreds of pollutants in samples containing thousands of poorly known natural compounds. The inability of the toxicologists to make sense of the tide of numbers we can so easily generate, and the frenzy of the press to misinterpret them are the only limiting factor to a tremendous growth.

Legal requirements regarding drug certifica- tion lead to a similar drive, one of the main differences being the terms in which cost and benefits are balanced, another being the need to prepare extremely pure compounds Ppharma- ceuticals”) and document their purity. The separation of complex mixtures and the identification of their components requires new progress in instrumentation, but mainly in the mere fundamentals of the methods.

These great promises could entirely vanish, however, if the regulations on the use of chemicals in research laboratories become too drastic, a distinct possibility at this time. In this case this would be the entire future of chemistry which would be in question. We have to be ready for a fight, even though we hope we will be spared.

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