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N e w chemical products and their application

Impact N o . 157 3 Editorial

Michael Freemantle

5 Porous or divided solids: a special state of matter Jean Rouquerol

17 The computer simulation of materials John Corish

27 Agrochemicals: pesticides and other strategies for pest control Roy Greenhalgh

37 Chemical drugs: new challenges Camille-Georges Wermuth

51 Strong fibres from flexible chain polymers Miroslav Raab

59 C F C s and their alternatives Michael Freemantle

71 Reference materials: their role in measurement accuracy W. P. Reed and S. D . Rasberry

81 Chemicals through biotechnology: facts, hopes, dreams and doubts Chandana Chakrabarti and Pushpa M . Bhargava

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Editorial

Chemical Abstracts Service (a Division of the American Chemical Society) currently registers over 600 000 new chemical substances each year and there are n o w almost ten million substances on their files. This compares with a cumulative total of approximately five million substances at the end of 1980 and about one million substances just over twenty years ago.

O f course, the vast majority of these substances are of academic interest only and unlikely to serve any useful purpose outside the research laboratory. Even so, an estimated 70000 chemicals are n o w in use in agriculture, consumer products and industry and this number is increasing at the rate of about 1000 each year. These chemicals serve to benefit society in a number of ways: food and drink, health, clothing, shelter, transportation and communication. Unfortunately, the large-scale use of some of these substances also causes problems—some on a global scale. In recent years the search for new, safer and therefore more socially and environmentally acceptable products has become urgent.

In this issue of Impact w e consider the development and applications of some of these new chemical products and materials. For example, Jean Rouquerol demonstrates the technological importance of porous solids in medicine, industry and environmental protection. In the following article, John Corish explains h o w simulation studies carried out on large, modern computers have m a d e an impact on our knowledge of new materials.

The fate of the environment and food safety are, as Roy Greenhalgh points out in his article, two aspects of science that are currently of concern to the public. H e presents an overview of pesticides and other strategies for pest control. Camille W e r m u t h reviews new medicinal chemicals including pharmaceuticals and Miroslav R a a b shows h o w new techniques for the effective control of the supermolecular structure of semicrystalline polymers have brought about dramatic changes in their mechanical characteristics.

Chlorofluorocarbons (CFCs), compounds which were once considered miraculous because of their stability, non-flammability and low toxicity, are n o w k n o w n to damage the earth's protective ozone layer. Michael Freemantle takes a look at materials that are being developed to replace these C F C s .

Reference materials have a wide variety of uses in science, industry and technology and also in environmental and clinical analysis, William Reed and Stanley Rasberry show h o w these materials are used to assure the accuracy and compatibility of measurements of physical and chemical properties in these various applications.

3 Impact of science on society, no. 157, 3-4

Finally, Chandana Chakrabarti and Pushpa Bhargava survey some of the n e w chemical products from biotechnology that are used in medicine, agriculture, energy, industry, animal husbandry and production, metallurgy, and pollution control.

In a single issue of a journal it is not possible to cover all the thousands of new chemical products and materials that are available to society. W e have had to be selective, and it has not been possible to include reviews on catalysts, electronic materials and other groups of materials of major social and economic importance. Nevertheless, w e hope that the following articles will indicate the essential contribution of chemistry, chemists and chemicals to the current and future welfare of our society.

Michael Freemantle

4

Porous or divided solids: a special state of matter

Jean Rouquerol

The porous or divided solid state enables the very numerous surface atoms to play a distinct role in the phenomenon of adsorption. This is observed in applications such as the purification and separation of gases and of liquids, the stabilization of suspensions like inks and paints, and in pharmaceutical or plant protection applications. The special problems presented by each of these cases and their present day solution are examined here.

W h a t do we call porous or divided solids?

W e are all familiar with the notion oí porosity, readily to be found in a sponge, in a piece of pumice stone and, on a m u c h finer scale, in the terracotta jug that is sufficiently water-permeable to allow cooling through evaporation from the outer surface. In some solids, the pores m a y be so fine that they only let through specific molecules, sometimes even specific atoms. T h e pore width thus covered ranges from a millimetre in the case of pumice stone d o w n to a nanometre (i.e. one millionth of a millimetre) in the modern "molecular sieve". Although the volume of these pores is limited (for the sake of rigidity of the structure, 1 c m 3 of porous solid "of technological interest" will usually contain no more than 0-2 to 0 6 c m 3 of porous volume), their walls are surprisingly extensive. The total area of these walls (called "surface area" even though w e are concerned with pores within a solid) m a y , in 1 gram of activated charcoal, attain over 2,000 m 2 . The properties of such a solid, so m a n y of whose atoms are in direct contact with the surrounding fluid m e d i u m , are understandably of a special character. In the example of charcoal, over 50 per cent of the atoms are thus only "skin deep" and capable of immediate interaction with a gas or a liquid. Figure 1 shows various aspects of the porosity of a solid that have a direct effect on its behaviour and on its technological usefulness, as will subsequently be illustrated with examples. A porous solid m a y

Jean Rouquerol is at present Director of the Thermodynamics and Microcalorimetry Centre of the National Scientific Research Centre ( C N R S ) , Marseille, which concentrates on the thermodynamic and kinetic study of solid or divided materials. H e is Research Director at the C N R S and has for thirty years been interested in the preparation (mainly thermal) and characterization (in particular by gaseous or liquid adsorption) of porous or divided materials, H e is Chairman of the Scientific Committee of the International Confederation for Thermal Analysis and, at the I U P A C , also chairs the Subcommission on the Characterization of Porous Solids and is a m e m b e r of the Colloid and Surface Chemistry Commission.

H e m a y be contacted at the following address: Centre de Thermodynamique et de Microcalorimétrie, C N R S , 26, rue du 141e R.I .A. , 13003 Marseille, France.


Jean Rouquerol

Figure 1.

Cross-section of a porous solid showing some of its features.

possess a shallow porosity called roughness (a), "bottle-shaped" pores (b), cylindrical pores (c), funnel-shaped pores (d), interconnecting pores (e), glove-finger pores (/) or completely closed pores (g). It m a y also possess slit-shaped pores, both wedge-shaped (like fissures) and with parallel walls. T h e "connectivity" (or degree of interconnection) of pores is a very noteworthy feature for rendering them rapidly available to a fluid. Recent studies have nevertheless shown that such availability m a y be totally blocked, well before the filling of the pores is completed in the course of the percolation phenomenon, by a sort of "traffic jam" in the porous network. It m a y sometimes suffice to fill the porous volume less than half full with a liquid (which in this case marks the "percolation threshold") to block any further passage of a gas. Flow forecasts are m a d e either with physical models (porosities "constructed" on a large enough scale to be visible) or through computer simulation. This outline affords a glimpse of the possible complexity of such a solid and the knowledge that has to be amassed in order to be capable of guiding its features in a given direction.

Divided solids are m a d e up of individual particles, and m a y cover a range of sizes almost as broad as that of porous solids. T o give an idea of these sizes let us say that they can resemble sand (i.e. grains of the order of a few tenths to one tenth of a millimetre), flour (of the order of a hundredth to a thousandth of a m m ) or an extremely light smoke (some particles of which m a y be as small as a few nanometres). Their surface area m a y range from a few hundredths of a square metre per gram (for a sand) to 200 m 2 per gram (in the case of silica smoke , referred to as "pyrogenic silica"). T o this variety of sizes m a y be added a great range of shapes, as suggested by the few examples in figure 2.

T h e tendency of a divided solid to agglomerate depends, in particular, on electrical interactions (between particles with surface electric charges), capillary interactions (in contact with a liquid or even simply a vapour) or universal-type interactions (known as "dispersion" interactions, not because they disperse solids—on the contrary, they have an attractive force—but because they disperse light). These interactions, as w e shall see,

6


Figure 2. Examples of divided solids, with the corresponding scales: (a) clusters of particles, 1 5 n m in diameter, of a pyrogenic silica of 200m 2 g - 1 ; ( b ) flat sheets in a kaolinite of 2 0 m 2 g ~ 1 ; (c) spheres of inorganic oxides of 0 - 5 m 2 g - 1 obtained by the sol-gel method or latex spheres obtained by polymerization in an emulsion; and (d) "acicular" crystals of magnetite, or of plaster, of 20 m 2 g" '.

need to be k n o w n and harnessed depending on the applications in view. Before

reviewing the latter, w e should point out that adsorption is involved in most

applications.

Adsorption

T h e word "adsorption" (yes, with a "d") was proposed early in the century by the American physical chemist Langmuir to denote absorption simply onto the surface, without penetration of the gas or liquid within the structure of the solid proper. The point is that the porous or divided solids just described are capable of concentrating and retaining at their surface considerable quantities of gases, vapours or liquids. They are called adsorbents. In fact, all solid surfaces adsorb (and even liquid surfaces too, but that lies outside our topic), though this effect is only noticeable and exploitable in the case of a highly porous or highly divided solid. For example, at ambient temperature and a relative humidity of 50 per cent, a small 1 c m cube of ordinary glass will adsorb

7

Jean Rouquerol

about two molecular layers of water, or approximately 0-3 of a microgram of water, which means an unnoticeable quantity. O n the other hand, a cube of microporous silica gel of the same size, whose porosity fills 30 per cent of its volume, adsorbs—even with ten times less humidity—300 milligrams of water, or one million times more, which becomes a matter of interest. In this example, the adsorption phenomenon amounts to condensing a vapour under pressure (e.g. a relative humidity of 5 per cent), at which normally it should not liquefy (a "saturating" vapour, i.e. with relative humidity at 100 per cent, is normally needed to produce condensation on the flat surface of a pane of glass, a bathroom tile or a saucepan lid). There in fact exists, in the immediate vicinity of the surface of a solid, an "adsorption field", with a universal attractive effect, which makes the difference. If a pore is sufficiently fine for the adsorbed molecules to be subjected simultaneously to the adsorption field of the two opposed walls (for which purpose the width of the pore must not exceed that of 4 or 5 molecules), the adsorption potentials are enhanced and the molecules are even more firmly retained in the pores, the latter becoming completely filled at still lower vapour pressures (sometimes as low as 1 per cent of saturated vapour density).

The adsorption of a vapour which condenses in this way in the immediate vicinity of the surface is called "physisorption". Figure 3 represents diagrammatically the four successive steps: adsorption first on the most active adsorption sites (I), which can, if needed, be measured in this way , although for this purpose it is preferable to use an adsorbable gas reacting chemically with these sites (which, according to the sites to be determined, will be ammonia , oxygen, hydrogen, carbon monoxide, etc.) and giving rise to what is called "chemisorption". There comes a time when the micropores (less than 2 n m in width) are filled with adsorbable gas and when the rest of the solid surface is covered with enough molecules to constitute, statistically, a dense monomolecular layer or "monolayer" all over the surface (II). The famous method of Brunauer, Emmet t and Teller (and k n o w n as the B E T method) makes it possible to identify this m o m e n t and determine the number of molecules adsorbed. F r o m the size of the molecule (usually the nitrogen molecule, calculated as covering an area of 0-162 of a square nanometre), the surface area of the adsorbent can be deduced. If the adsorbable vapour pressure continues to be increased, w e arrive at the stage represented in III: some pores (a and b) are completely filled, others (c) partially so through "capillary condensation", and others, still larger (d), are only lined with a layer of the same thickness as the parts of the external surface (e). By increasing the pressure still further, w e ultimately fill all the pores, even the widest (IV). F rom Kelvin's L a w it follows that to each adsorbable gas pressure corresponds only one single pore size being filled (or emptied, depending which way the pressure is being varied). This has given rise to an elegant method (the Barrett, Joyner and Halenda method, also k n o w n as the B J H method) for establishing the pore-width distribution of "mesopores", which are as little as 2 to 50 nanometres wide, without electron microscopy (which incidentally does enable a three-dimensional picture of the porous network within a material to be easily built up).

Technical applications and new applications of porous or divided solids

The last half-century has seen a considerable research effort into what is commonly called the "physical chemistry of interfaces" or the "physical chemistry of colloids" (a

8


Figure 3. Successive steps of the adsorption of a gas or of a v a p o u r b y a porous solid. I. Site (or "localized") adsorption. II. Adsorption in statistical m o n o l a y e r a n d in a "micropore" (a). III. Adsorpt ion in multilayer a n d capillary condensation in particular "mesopores" (b a n d c). IV. Saturation by an adsorbed vapour in a state comparable to the liquid state.

colloid being definable in very general terms as a substance displaying size differences in the range of a thousandth to a millionth of a millimetre, i.e. between a micrometre and a nanometre, which comprises, in particular, most of the porous solids to which reference is m a d e here). As a result, m u c h has been learned about the behaviour of these materials in the presence of gas or liquid, and w e very often k n o w nowadays h o w to prepare them by finely orienting their properties in the direction of the desired applications. S o m e of these will n o w be described, with emphasis on the special requirements that had to be met in each case.

9

Jean Rouquerol

Clear liquids

The purification of liquids is the prime use of the adsorbent most widely manufactured today in the world, "activated charcoal" (i.e. porous charcoal).

For example, the drinking water of most major cities completes its treatment with a "refining" in which it flows through an activated charcoal bed designed to trap definitively every kind of residual chemical pollutant that the previous steps in the treatment have been unable to eliminate completely from the river water often used: chlorinated solvents, detergents (also called "surfactants"), and products of the decomposition of plant matter in marshes (i.e. fulvic or humic acids). The relative molar masses of these substances range from 50 to 15,000 and their molecular sizes are in m u c h the same ratio, which demands a very wide pore-size distribution of the charcoal used. In effect, the molecule is retained all the better if it finds a pore of its o w n size. This rules out a charcoal possessing only wide pores; not only would the small molecules be weakly adsorbed but, in addition, the surface area of this charcoal would be low, as would its adsorption capacity. Today we are able to m a k e activated charcoals containing all pore sizes between 0-3 n m and 50 n m simultaneously. W e are able at the same time to give them organophilic properties (thus enhancing adsorption of the organic pollutants just mentioned) and hydrophilic properties (conducive to good wetting right to the bottom of the pores). W e k n o w h o w to obtain them from coal, peat or w o o d charcoal (of beech or pine). W e k n o w h o w to "activate" them (i.e. "open" their porosity) by subjecting them to steam, carbon dioxide or phosphoric acid, and w e k n o w h o w to convert them either into rigid grains able to withstand handling, use in a "flow-through bed" and an annual or two-yearly regeneration, or there again into single-use fine powder that can be eliminated after décantation.

Similar adsorbents are used to decolourize the juice of sugarcane ("molasses") and edible oils, or to m a k e white vermouths. The innocuous nature of these adsorbents is such that, as we shall be seeing, they can also have pharmaceutical uses.

W h e n conversely, in the field of fine chemistry, the requirement is to eliminate the last traces of water present in an organic liquid, use is also m a d e of an adsorbent such as a "molecular sieve" of the 4 A type, whose 'windows' will admit only the water molecule, which is particularly small. This adsorbent is a highly preferable substitute for metallic sodium, which had to be press-extruded in the form of a strip and immediately immersed in the liquid, an operation that was not free of the risk of explosion when a solvent with high vapour pressure, like acetone or ether, was being dried.

Adsorption at the solid/liquid interface is also the principle used in stain-removing compounds for clothes. The colour-producing molecules of the stain are dissolved by an aromatic solvent and diffuse within it towards very fine grains of a ground silica gel on which they are adsorbed. The solvent evaporates and the dry adsorbent (still white because adsorption takes place in the pores, within each particle) can simply be brushed away.

Inks which do not settle

Once again it is carbon, but this time in divided (and no longer porous) form, that is still the most widely used pigment today, followed by the white pigments zinc oxide and, in particular, titanium dioxide. The predominance of carbon is due to the fact that it is used not only in paints but, above all, in printing inks and in all modern plain-paper

10


photocopying machines. This divided carbon (known as "carbon black", and which is really a smoke black obtained through the incomplete combustion of hydrocarbons) is produced, if needed, in sizes as small as 30 nanometres, giving it in such a case a surface area of about 100 m 2 per gram.

T h e main problem to be overcome is that of the stability of suspensions. The Brownian motion to which these particles are subjected (and which is the result of unequal shocks received from the molecules of liquid themselves subjected to thermal agitation) tends to bring them into collision and the very short-range universal interactions (particularly the "dispersion forces" referred to earlier and which have a range of a few atomic dimensions only) then keep them agglomerated. T o keep the particles in suspension one can act upon the electric charge of the particles (since two similarly charged particles repel each other) and this depends directly on the acidity or basicity of the surrounding aqueous solution (measured in terms of p H ) .

Another elegant solution is to adsorb onto the particles molecules that are long enough to keep them further apart than the range of the dispersion forces. These adsorbed molecules are usually macromolecules, and m a y be of plant origin, like the g u m s which were used to stabilize Indian ink long before their action was understood. This method is called "steric", for it operates on the size of the molecules. It is particularly suitable when the pigment is in suspension in an organic liquid (as in the case of oil paints and gloss paints).

Non-drip paints

W e m o v e on from the area of the stability of suspensions to that of their consistency, their viscosity, their elasticity, and possibly even their rigidity which m a y exist up to a certain point, as in the well-known example of quicksands. These features enter into what is more generally referred to as the "thixotropic" behaviour of suspensions. They are particularly important for thick liquids and pastes.

T h e problem sometimes consists of obtaining as concentrated a solution as possible while maintaining a viscosity suitable for ease of pumping: for example the lubrication of boring bits with finely formulated "drilling muds", the feeding of boilers with thick suspensions of powdered carbon, or the coating or "surfacing" of paper by means of a suspension highly charged with kaolinite (over 60 per cent) and yet needing to be capable of being spread thinly over the surface of an uninterrupted sheet moving at high speed towards the dryer and the reel. It is also, of course, true of the making of "technical" ceramics by slip casting in plaster moulds of complex shapes whose porosity immediately ensures, by capillary suction, the partial drying of the slip. The adsorption of surfactant molecules onto solid particles and sometimes also the control of their electric charge provide the necessary internal lubrication.

O n other occasions, the problem is h o w to obtain a sort of semi-rigid structure with the aid of the solid components of the suspension, which then need to be in mutual contact to constitute a kind of scaffolding that m a y then be destroyed at low cost, e.g. by agitation using very little energy. This "temporary construction" effect is obtained in a somewhat spectacular fashion with pyrogenic silicas whose particles (15 nanometres in diameter in the example represented in figure 2 a) link up spontaneously (as a result of their surface hydroxyl functions giving rise to weak bonds called "hydrogen bonds") in fine clusters to form a veritable net enveloping and passing through the liquid in which they are placed in suspension. With a content of just a few percent w e obtain the

11

Jean Rouquerol

Figure 4.

Scaffolding of kaolinite crystals under the effect of electric charges in an acid medium.

appearance of a gel-like material with petrol (forming the notorious napalm) or with paint, office glue or neoprene glue, products which are often marked " N e w , non-drip!".

Kaolinite is also a useful thixotropic agent. W h e n positively charged (which happens in an acid medium), the edges of its flat crystals stick together at right angles to the basal faces of the neighbouring crystals (which, for their part, bear a permanently negative electric charge). The result throughout the suspension is a "house of cards" structure (see figure 4), which collapses immediately on switching to a basic medium.

The size and the shape of the accumulations or "agglomerates" of particles is, of course, the first information w e need in order to understand and control these stiffening phenomena. Until recently all the available methods for grain-sizing were applicable only to diluted suspensions and left it wide open as to what happened in a concentrated suspension. This problem has n o w been overcome by means of small-angle neutron scattering, an expensive but in some cases irreplaceable technique.

Pure gases

The separation of gases is a major application of adsorption, and firefighters' gas masks are a familiar example of this. These masks used to be highly uncomfortable for the wearer because of the effort needed to suck air through the activated-charcoal bed. The problem has n o w been resolved by means of a textile activated charcoal (obtained by the carbonization and activation of fabrics woven from organic synthetic fibres), making breathing ten times easier. Activated-charcoal filters are found in all devices used for recycling breathable air (whether in submarines or in kitchen cooker hoods). Preference goes then to charcoals with slit-shaped pores (which are the most numerous since this sheet-like graphite structure is engendered, even in embryo fashion, by carbonization) capable of very high adsorption of the flat molecules of carbon dioxide and of aromatic organic substances.

The prevention of atmospheric pollution by factories is also dependent upon adsorption. A critical example is that of aluminium production plants. Aluminium is

12


obtained by the electrolysis of alumina, itself dissolved, at less than 800°C, in a melting med ium very rich in fluorine, cryolite (in its pure state, alumina would melt only above 2,050°C). The escaping fluorine vapour used to fall on nearby grazing land and give cattle fluorosis, too m u c h fluorine being scarcely better for teeth than none at all. The problem was overcome by supplying the factories in part with a special-grade alumina in porous form, whereas traditionally use had been m a d e only of a non-porous a-alumina. This is first placed in air filters where it adsorbs the fluorine vapour before being incorporated in the cryolite bath to which it restores the lost fluorine; in this way, everyone profits. The porous alumina is prepared by thermal dehydration of an aluminium hydroxide. The loss of the water molecules leaves micropores in place (the width of a molecule) which m a y be widened at will in the subsequent thermal treatment. This type of study gains m u c h from the scope n o w offered by controlled transformation rate thermal treatment, which permits the precise selection of test conditions.

The separation and concentration of important industrial gases (such as nitrogen, oxygen, carbon dioxide, etc.) is also n o w carried out by adsorption, the sole previous practice for a long time having been liquefied gas distillation, which consumed far more energy and material. The present methods use compression-adsorption and decompression-desorption cycles (pressure swing adsorption) accompanied by coolings and heatings regarded as economically acceptable. Devices of this type are even used at sea to remove nitrogen from the helium and oxygen mixtures breathed by divers working on offshore oil installations. Even if the pressure of this gaseous mixture attains 40 bars (for a diver working 400 metres down) the partial pressure of nitrogen must remain well below 5 bars to prevent the hallucination k n o w n as "depth intoxication", which takes away the diver's will to surface again.

Dehydration of the air is also an important application where silica gels, whose hydroxylated surface is highly absorbent, come into their o w n . Obtained by the precipitation of silicic acids in the form of a loose agglomerate of particles ("gel") they are then over-dried to yield a hard, vitreous-looking "xerogel" that retains a major part of the porosity of the initial hydrated gel. Precipitation and dehydration can be controlled so as to obtain all sorts of pore sizes (between 0 5 and 100 nanometres) and a whole range of apparent densities (between 0-1 and 2). The lowest densities (those of the "aerogels") are, for example, obtained by temporarily replacing the water with an organic solvent possessing a lower surface tension (such as alcohol, acetone, etc.), whose loss in the drying process causes less shrinkage of the porous structure than in the presence of water.

W e are also able to m a k e , for the electronics industry, porous glass parts serving both as insulators and as dehydrating adsorbents. Once encapsulated with the components, these parts ensure the lifelong low humidity the components need. These porous glasses are obtained from a diphasé glass, one of whose two phases, initially distributed spiderlike through the other which is m a d e of pure silica, is dissolved in an acid bath. With this method particularly uniform pore sizes can be obtained. The mechanical stability and excellent resistance to aging of these solids has led to their being chosen as reference porous adsorbents by the Communi ty Bureau of Reference ( B C R ) in Brussels.

Finally, molecular sieves are used to ensure, invisibly since they are concealed into the edging, the permanent dehydration and hence lack of condensation in double-glazing components.

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Jean Rouquerol

Hundreds of other applications each with their own requirements

There are in fact so m a n y fields of application for porous or divided solids that w e have to m a k e do here with a partial list.

In pharmacy, for example, w e can cite the gastric adsorbents (activated charcoal, montmorillonite, alumina or magnesia) for combating heartburn or certain forms of poisoning. Textile-activated charcoal is also beginning to be used as an adsorbing and, of course, completely sterile dressing for serious wounds or burns. Furthermore, although the pharmacist has traditionally been accustomed to grinding powders in his o w n mortar, our requirements have become more and more specific. In come cases, the tablet needs to disperse and dissolve immediately in water; in others, by contrast, it must dissolve slowly in the gastric juices so as to release the active substance only gradually and maintain a constant concentration in the patient between two doses. This is achieved by a simultaneous control of grain-size and the surface state of the powder, particularly by adsorption.

Automatic blood-cell counting devices are calibrated by means of suspensions of latex spheres in the required size range (2 to 10/mi). Their exceptionally uniform grain size is obtained by dispersing in a liquid droplets of m o n o m e r that are subsequently polymerized.

The plant protection industry also makes extensive use of powders that must not become agglomerated (or form "clumps") or ultimately alter their grain size, for their activity is directly dependent upon the finest and most uniform possible dispersion over the entire plant. Yet some of these powders are liable to low-temperature sintering, a case in point being "flowers of sulphur". Although it does not melt until about 115°C, this sulphur (principally in the form k n o w n as a) is capable of sintering from 50°C upwards, a temperature possible in a shed or in a sack exposed to direct sunlight, which means that its particles stick together and coalesce, gradually melting into one another (see figure 5). Other powders undergo a so-called "Ostwald ripening" in the course of which the finest grains (unfortunately accounting for the bulk of the surface area) disappear to make w a y for the largest (see figure 6). This phenomenon, which can take place in the presence both of air and of liquid, is due to the greater vapour pressure (or greater solubility) of the particles with the smallest radius of curvature. This is nowadays avoided either by preparing powders of a single particle size or by coating them in a protective adsorption layer, which, where necessary, also retards sintering. It is often profitable to adsorb for this purpose a surfactant (e.g. a lignosulphonate, an inexpensive by-product of the paper industry), which, in addition, assists dispersion of the aqueous solution at the time of use.

Figure 5.

Coalescence of two grains during sintering at a temperature well below melting-point.

0 = 0 14


O o

o O

O O

° O

o

O o °

O o O

O

=̂ >

ü

O

o

Figure 6. Disappearance of the finest grains by "Ostwald ripening'.

The field of hydraulic bonding agents, such as cements and plasters, is par excellence the field of the divided states (before use) and subsequently porous states (after setting). Although these binders harden not by drying but by hydration, m a x i m u m resistance of the concrete is obtained by mixing with a m i n i m u m of water, which sometimes makes it necessary to add fluidizers (polymers and surfactants). O n c e it has set, the cement paste is a microporous m e d i u m that remains essentially filled with water. Being perfectly suited to water, which has produced them as it were, the micropores are mostly unavailable to the nitrogen molecule since it is larger (it is usually reckoned that the water molecule covers an area of 0-105 n m 2 , as against the 0-162 n m 2 for the nitrogen molecule). The surface area available to water and determined by the B E T method exceeds 500 ^ m 2 per gram. Curiously enough, these micropores gorged with water present no danger in the event of frost, since the strong interaction of the water molecules with the walls completely prevents them from organizing themselves with the pattern of the ice network. O n the other hand, even though far less numerous, the fissures wider than 0-1 /xm in which ice can crystallize, are really the weak point of this material. Hence the recently exploited idea of sharing the shrinkage between a greater number of narrower but not frost-prone fissures.

T o conclude without truly closing the subject, suffice it to view at our feet the inexhaustible supply of porous or divided solids provided by the soils—themselves the subject of an entire branch of science—in order to reflect upon the importance and universality of this state of matter. •

Bibliography

For a general introduction to the physical chemistry of colloids: E V E R E T T , D . H . (1988) Basic principles of colloid science. Royal Society of Chemistry Paperbacks, 243 pp.

For an introduction to characterizations by adsorption: G R E G G , S. J. and S I N G , K . S. W . (1982) Adsorption, Surface Area and Porosity. Academic Press, London, 303 pp.

For an introduction to methods of separation by adsorption: R U T H V E N , D . M . (1984) Principles of adsorption and adsorption processes. John Wiley and Sons, N e w York, 433 pp.

15

The computer simulation of materials

John Corish

The use of computers to model real-life situations now includes the design, on an atomic or molecular level, of specialized materials such as ceramics, alloys, components for electronic devices and pharmaceuticals. The positions and movements of individual atoms are modelled in powerful computers which then interpret their behaviour to predict the relevant properties of the materials they constitute. Ultimately these techniques have the potential to replace some of the expensive speculative work which currently has to be done in the laboratory on materials synthesis and testing.

T w o of the most striking changes that have taken place in science and engineering in the last decade have been the widespread availability of large, fast computers and the emergence of an exciting range of n e w materials designed and m a d e with specific properties to fill well-defined needs in the marketplace. It will hardly surprise anyone to learn that these two developments are not completely unrelated. In fact, the use of computers to simulate the behaviour of n e w materials is n o w a c o m m o n and increasingly important technique in their development. W h e n materials are already in existence the simulation of their properties greatly increases our understanding of their nature and of the forces that operate between their constituent atoms. A s an extension of this kind of study, computer simulation can be used to predict the likely properties of modified or even entirely novel materials before the difficult, exhaustive and often expensive step of trying to m a k e them in the laboratory is undertaken. Although the use of computer simulations encompasses gases, fluids, polymers and solids w e will concentrate here on solid materials, where the technique has m a d e major contributions in both academic and industrial contexts. This type of simulation typically requires very large and fast computers, usually n o w referred to as supercomputers, and it is the emergence of such machines that has m a d e realistic calculations feasible. However, the simulations also rely on the great wealth of fundamental physics, chemistry and

John Corish is Professor of Physical Chemistry at Trinity College, University of Dublin. A graduate of the National University of Ireland, he has also taught at the University of Western Ontario, Canada and at the University College, Dublin. His research interests include both theoretical and experimental studies of matter transport in a wide range of solid materials. H e has worked on simple ionic materials, on the high-temperature gaseous corrosion of superalloys, on advanced polymer-based battery systems and, most recently, on the transdermal delivery of drug molecules. H e is President of the Institute of Chemistry of Ireland and Secretary of the I U P A C Commission on High Temperatures and Solid-State Chemistry. H e m a y be contacted at the Department of Chemistry, Trinity College, Dublin 2, Ireland.


J. Corish

mathematics that has been painstakingly assembled over m a n y years and which provides the equations necessary to calculate bulk and other relevant properties of a material from a knowledge of the forces between its atoms.

This article will describe the ways in which the ability of the enormously powerful supercomputers capable of carrying out a very large number of operations simultaneously, have been used to simulate the behaviour of individual constituent atoms in materials. Then h o w , by combining the results of m a n y such related calculations, an overall picture can be built up to show h o w the bulk macroscopic material behaves. W e shall also see h o w modern computer graphics coupled with software and hardware developments have revolutionized the work of the synthetic scientist w h o is seeking to m a k e novel molecules in the laboratory to fit precise requirements. The new techniques can eliminate m a n y hours of costly experimental work by clearly defining the types of molecules that will eventually be suitable for the task in hand.

Atomistic simulation—crystalline materials

It is a central tenet of science that all materials are composed of atoms. These are extremely small particles of matter whose sizes are very far below our normal range of comprehension. It follows naturally that it takes m a n y millions of millions of these atoms to form a piece of matter that w e can see and whose properties w e can easily recognize and measure. In crystalline solids the atoms are organized into repeating patterns in three dimensions and the symmetry of these lattices provides an important key to our ability to simulate their structure and properties. Different, and often less precise methods must be used for non-crystalline amorphous materials. The principal characteristic of computational calculations is that a very large number of repetitive operations can be executed quickly and without the errors that would be almost inevitable if time permitted such work to be done by h u m a n beings. The connection between the facility provided by modern fast computers and the needs of atomistic simulation calculations are obvious. Techiques have been developed to compensate for the fact that the capacity of even the largest computer cannot provide sufficient space to simulate all of the atoms in even a very tiny piece of material on an individual basis. Supercomputers, because they often use array processors in which m a n y operations are carried out at the same time, are particularly suited to the type of calculations required.

The crucial function in the simulation process is the transformation of information relating to the microscopic behaviour of individual atoms into useful macroscopic properties that describe the bulk behaviour of the material. There are two requirements for this transformation: the first is that the forces operating between the individual atoms can be described in a usable mathematical expression; the second is that the necessary formalism exists to transform the calculations on individual pairs or small groups of atoms into results for macroscopic properties that refer to usefully-sized pieces of the substance.

Fortunately, the study of the forces between atoms has always been of fundamental importance and interest to scientists w h o long ago devised equations to express their variation with the distance of separation of the species for both short-range and long-range interactions. These potentials vary from rather simple expressions in which the atoms are treated as hard spheres to very sophisticated theories that take full account of

18


the electronic structure of the atom and its effects on interatomic interactions. Fortunately also, the formalisms of lattice dynamics provide the equations to calculate properties such as the elastic constants of a substance from a knowledge of the forces between the individual atoms that m a k e u p its crystal structure. Similarly the dielectric properties, the lattice or cohesive energy of the crystal as well as any strains that m a y exist in its structure can be evaluated. Although some of these calculations were considered as major tasks and took very lengthy times to accomplish before the availability of modern computers, they can n o w all be completed, even for the most complex materials, in times of the order of seconds.

So h o w is the simulation carried out? T h e most powerful and detailed modelling is of static lattices in which the thermal vibrations of the atoms are ignored. The programs, once given a description of a small part of the crystal, use clever and versatile symmetry packages to construct the complete lattice from this information. The specified potentials are used to calculate the energy of the crystal. T h e most likely structure is then predicted by adjusting the positions of the atoms and the lengths and angles of the chemical bonds between them to minimize the energy of the system. W h a t this means is that the components of the crystal lattice are allowed to m o v e about in the computer in a fashion that causes the energy of the system to run 'downhill' and to assume the lowest energy configuration. This is just what would be expected to happen in nature as the atoms seek to find their most comfortable position in the real material. Very effective routines to accomplish this energy minimization have been devised and because these do not require very m u c h space in the computer processor it is possible to use quite sophisticated potentials to model the forces between the atoms. This in turn means that, within the limitations of the omission of atomic vibrations and time-dependent phenomena, very accurate results for the atomic arrangements of the atoms in the crystal can be obtained and that the calculated estimates of the energies are found to be very accurate indeed in cases where they can be compared to experiment.

Application to defects and surfaces

Perhaps the most important advantage of these energy minimization techniques is their ability to calculate the energies and structures of point defects in solids and of solid surfaces. T o a solid-state scientist, surfaces, because they break the regular arrays of atoms of the material in which he is interested, appear as defects. Together with point defects, which are atomic scale irregularities such as vacant sites or additional interstitial atoms that are squeezed into a structure, surfaces provide the most active sites in the material and those at which chemical processes are most likely to occur. In fact, the transport of matter through solids by diffusion can generally only take place because of the existence of such lattice imperfections. This is because, unlike liquids and gases in which the atoms are free to m o v e about, the atoms in solids are fixed in positions and, under normal circumstances, they only undergo vibrations about these sites. Diffusion processes through solids, occurring via vacancies and interstitials, are responsible for a variety of phenomena such as the corrosion of metals and alloys, the operation of numerous advanced solid-state battery systems and the transdermal delivery of drug molecules from polymer reservoirs. Thus the application of static lattice techniques to model the creation of these defects and to calculate the energies required to promote defect processes such as diffusion is vitally important in the

19

J. Corish

C-axis

Fe3

(o) O Fe3+ interstitial

Energy (kjmol1)

300

200

100

(b)

Figure 1.

The modelling of diffusion.

(a) The iron ion, A , is situated in an interstitial site in the oxide crystal. This is a naturally occurring defect and if the ion is given sufficient thermal energy it can approach the iron ion B . Diffusion occurs w h e n ion B is knocked from its lattice position to another interstitial site C . Ion A then occupies the vacated lattice site at B .

(b) A s well as providing this extremely detailed mechanistic picture of ionic diffusion the simulation also calculates the energy necessary to cause the m o v e m e n t to happen. T h e curve shown here is constructed from a series of calculations in each of which ion A is m o v e d progressively closer to the lattice ion B that it displaces. T h e energy profile shows that the concerted movement occurs w h e n ion A has m o v e d approximately one third of the distance towards the lattice ion.

20


Ideal

(ic

t O

® o o

surface

2 o o i> & o O Q

^

o o o @ © o o •

'ositive ion

Rumpled surface

Q Surface ̂ O ^

® o ® o o # o ^ ^ o ^ o o ^ o ^

O

o ^

o

(__) Negative ion

o ® 0 O O 0

• o

Figure 2. The rumpling effect shown by simulation to be present on the surfaces of ionic crystals such as on the (100) surface of sodium chloride (common salt), where the sodium ion is the positive cation and the chloride ion the negative anion.

development of n e w commercially attractive materials. T h e detailed modelling of the m o v e m e n t of an iron ion through iron oxide, which is an important component of m a n y of the corrosion reactions causing so m u c h destruction of our heavy industrial plants, is illustrated in figure 1.

All because of their potential applications the techniques have been extended to model surfaces where they can also provide a remarkably detailed picture of the structure and energetics of the atoms in the layers near to the surface of the crystal. For a start, they have shown that these surfaces of ionic crystals are not completely regular, as might have been assumed, but as s h o w n in figure 2 present a rumpled appearance, with the negative ions protruding slightly above the ideal plane. They also predict the ways in which different types of impurity ions can be expected to segregate preferentially towards surfaces. This type of information can be crucial in our understanding and development of n e w catalytic materials which are the key to so m a n y industrial processes. Indeed, it is the facility in the modelling techniques to change from one dopant or impurity ion to another, by simply changing the relevant interatomic potential, that confers o n them their great advantage. It is m u c h easier, faster and less expensive to simulate a novel idea in this w a y than it is to m a k e the material in the laboratory and to then test it experimentally. For example, the modelling of changes in minerals that are normally related to geological timescales can be carried out in an afternoon.

Since it is also possible to simulate the movement of atoms or ions through lattices by diffusion, or under the influence of an electric field, it is also feasible to assess various dopants, both qualitatively and quantitatively, for their ability to influence the transport properties of a host crystal. In this area computer simulation has been used to interpret the effect of the addition of calcium ions to zirconia to produce the calcia stabilized material which is a most important commercial oxygen ion conductor. This

21

J. Corish

Figure 3.

The simulation of a conducting polymer.

(a) This shows the square channels, looking end on, formed by polyacetylene chains with potassium dopant ions on their central axes and is the configuration supplied to the computer before the relaxation calculation.

(b) Here three potassium ions are viewed sideways-on with only two of the polyacetylene chains that form the channel which they occupy shown. The calculations reveal the optimal positions for these ions as well as the buckling effect which they impose on the polymer chains. T h e simulation can also be used to show that the potassium ions are highly mobile along the channels.

22


ceramic is vital to the operation of the oxygen gauges that are used to ensure complete and clean combustion in large commercial furnaces and in combustion engines. The motion of dopant ions into polymers, a process which gives rise to the production of the exciting range of new materials k n o w n as conducting polymers, has also been investigated by computer modelling. Figure 3 shows h o w the dopant ions are accommodated between the polymer chains in polyacetylene, a prototype conducting polymer.

The very initial stages of the aggregation of impurities and defects in materials have also been simulated successfully. Information of this type is important for two reasons. Firstly, it is necessary to understand the processes concerned, since they are essential for the formation and separation of separated new phases that can completely change the properties of a material. Secondly, it is usually very difficult to obtain the information in any other way, because the sizes of the collections of atoms involved m a k e them too complicated to be understood on the basis of measurement techniques suited to individual atoms, while they are too small to be seen experimentally by the usual structural determination methods. Simulations of this type of cluster of atoms have recently provided essential background for the interpretation of a variety of experimental structural data.

Modelling dynamic processes

For m a n y applications it is necessary to include the atomic kinetic energy explicitly in the modelling. This can be done using the techniques of molecular dynamics. Here the principle is quite simple in that a number of particles are put into a box in the computer's m e m o r y , each is given a position and velocity and, as before, the interatomic forces acting between them are specified in terms of a potential. The simulation is m a d e by allowing the ensemble to pass through a succession of time steps with Newton's equation of motion being solved each time for the components. These time steps are extremely short, since they have to correspond to motions on an atomic scale, and several thousands of them must be allowed to take place in each simulation. The simulation box is first set up by specifying the contents and assigning velocities corresponding to the required temperature. The ensemble is then allowed to equilibrate so that the kinetic and potential energies of the particles are correctly partitioned and a thermalized distribution of velocities is attained. There then follows a production run in which the behaviour of the particles, positions and velocities, are recorded at each time step in a manner that allows the data to be analysed later.

The mathematical procedures that have been developed to control the generation of information in a molecular dynamics simulation and to interpret the data are complex. For our present purposes it is sufficient to recognize that the technique can provide an almost exhaustive amount of information on the ways in which atoms and molecules organize themselves to form liquids and solids and, in particular, h o w particular particles m o v e by diffusion through a material. The technique provides a series of snapshots of the substance that can be analysed in detail to follow the trajectories of individual atoms. This means that detailed mechanisms for diffusion can be identified, and the technique has, in this way, added greatly to our understanding of m a n y phenomena that occur in solid materials.

Molecular dynamics is the most versatile and probably the most widely used technique for the simulation of materials. It is, however, very demanding on computer

23

J. Corish

time and m e m o r y and, as a consequence, the level of sophistication of the interatomic potential that can be used is often less than ideal, such that the accuracy and reliability of the results are reduced. Even with the most modern powerful computers it is sometimes also difficult to include a sufficient number of time steps in the simulation to ensure that the process of interest takes place to a statistically significant extent within the sampling period. Nonetheless, the technique is continually being developed and extended and can only become more important in the future as available computational power increases.

Simulating molecules—molecular graphics

Another area in which computational techniques have m a d e a major impact on the development of new materials is in the design of molecules, particularly for biological and pharmaceutical applications. Here the uniqueness of the contribution lies in software and hardware developments that allow the production of an accurate three-dimensional image of even the most complex molecule, which can be displayed on a screen and rotated so that the scientist can view it from any direction. In its most simple m o d e molecular graphics can be used to model a molecule whose structure is already k n o w n from X-ray crystallography or other techniques for structure determination. M o r e valuable, however, is the ability of the simulation to use energy minimization techniques to predict the overall shape of a suggested molecule, including different configurations which it m a y adopt or through which it m a y pass. In m a n y modern pharmaceutical applications new products are required in which the ability of the molecules to selectively interact with, and bind to a specific site on a biological substrate is essential. If both the receptor site and the potentially active molecule are simulated then the synthetic chemist can examine them together to see if their shapes and sizes are such that they can be expected to interact. H e can also examine possible alternative molecules that, while retaining the necessary pharmaceutical activity, would be more likely to attach themselves to the target site. Ultimately, he can design a molecule with the necessary characteristics for the specific application, and the computational systems n o w available provide him with a choice of display styles that enable him to accurately test its suitability to bind to and interact with the receptor site.

The use of molecular graphics is not confined to developments in the pharmaceutical industry. The techniques can be used to model, display and investigate modifications to any molecular system. They are particularly well suited to systems in which the relative sizes of interacting species are important, such as selective adsorption in catalysis, and to the design of artificial enzymes and the development of molecular devices, and are n o w an essential tool for the synthetic chemist. They ensure that his experimental endeavours are directed at making molecules and materials that have a real change to succeed in the task for which they are designed. In this way they provide a highly sophisticated three-dimensional drawing board for designing molecules in his work as a molecular engineer.

The future

Scientists have responded extremely effectively to the very rapid increase in the speed and power of modern computers and their more ready availability, and have used them

24


to increase our understanding of materials. The obvious correlation between the ability of the large array processor to handle m a n y pieces of information simultaneously, and the frenzied activity that is typical of the atoms or molecules in a piece of material has been fully and vigorously exploited. The range of techniques developed n o w covers almost all aspects of the science of materials and the subject is increasing in importance as a predictive as well as an investigative facility. There remains a very significant difference between the numbers of calculations that can be accomplished, even in the largest computers, and the numbers that are ideally required in the simulation procedures, although this gap will be progressively closed as more powerful machines are built. At present, it is the quality of the interatomic potential that can be used that largely determines the quality of the result and again this is dependent on the computational power that is available. M o r e powerful machines will m e a n that more realistic models can be used and that the techniques will yield more accurate results and can be extended to more complex materials.

Perhaps the ultimate achievement might be seen as a program which, given the relative numbers of the different atoms in a substance and accurate potentials for the interactions between them, could predict the structure and properties of the material that would form. This is, however, hardly ever likely to be realized or to be necessary because the scientist, n o w as always, uses a logical approach that seeks to build on the knowledge that he already has. Thus he will always use his experience to the full in the early stages when the simulation process is being set up. Programs already exist that can, if the calculated energy profile suggests it, change an initially proposed crystal structure to a more likely configuration. This type of information is extremely useful in the investigation of phase changes and will become more important as the variety and quality of the specialist materials that are produced in response to market needs increase. The demand for these materials, which is spread throughout all areas of science and engineering, will ensure that the atomistic simulation techniques discussed here, static lattice energy minimization, molecular dynamics and molecular graphics, will become even more versatile and powerful and that they will be m o r e widely used in the future. •

25

Agrochemicals: pesticides and other strategies for pest control

Roy Greenhalgh

Pesticides have made a positive contribution to our lifestyle by reducing diseases and increasing food supplies throughout the world. Concern for the fate of the environment and the safety of foods, however, has led to the search for novel strategies for pest control. The biorational approach to the design of new pesticides ensures specificity of action and compatibility with the environment. Interest in biological control is increasing as an alternative strategy, but chemical pesticides will continue to be an important component of any pest management system. It is biotechnology, and our ability to manipulate organisms genetically, that hold the greatest potential for providing novel approaches in pest control and improving the quality of the environment.

Regardless of which country w e live in, our lives are affected by some aspect of science. Advances in medicine have led to longer lifespans and the food w e consume is more nutritious, providing a healthier lifestyle. Despite these benefits, two aspects of science are currently of concern to the public, namely the fate of the environment and food safety. Agrochemicals, by the nature of their use, have impact on both these concerns.

The term agrochemicals covers m a n y compounds but here w e will be concerned only with pesticides. This latter term is generic and includes insecticides, herbicides and fungicides which control pests, i.e. insects, weeds and fungal pathogens, that affect our health, environment and food. Although m a n y naturally occurring compounds were used as pesticides in the past, e.g. sulphur, nicotine and Bordeaux Mixture (an aqueous solution of copper sulphate), it w a s the advent of the synthetic organochlorine compounds that ushered in the era of pesticides as w e k n o w them today.

Public concern over the environment and the use of pesticides first arose following the publication of Silent Spring by Rachel Carson in 1962. However, it is only recently that regulations have been implemented with regard to the environment. In contrast, the safety of food has been regulated for decades, both nationally and internationally.

Dr. Roy Greenhalgh is a Principal Research Scientist with Agriculture Canada and an Adjunct Professor at Ottawa and Carleton Universities in Ottawa. A n organic chemist by training, his research has covered a wide range of topics with m a n y publications in the fields of organophosphorus, natural products and pesticide chemistry. Currently, he is studying the chemistry of Fusarium mycotoxins. H e is a m e m b e r of the I U P A C Commission on Agrochemicals and is currently secretary of the I U P A C Applied Chemistry Division. In addition, he has served as an F A O expert at J M P R (Joint F A O / W H O Meeting on Pesticide Residues).

Dr. Greenhalgh m a y be contacted at the following address: Plant Research Centre, Agriculture Canada, Ottawa, O n , K.1A 0C6, Canada.


R. Greenhalgh

Codex Alimentarius, which operates under the aegis of the U N / F A O and U N / W H O consists of the Codex Committee on Pesticide Residues ( C C P R ) and an F A O / W H O expert Joint Meeting on Pesticide Residues ( J M P R ) . The latter body serves to establish permissible levels of pesticide residues in agricultural products for international trade. In addition, most countries have their o w n agencies that set national permissible levels of residues in agricultural products and ensure that the consumption of food will be safe. In the U S A , it is the Food and Drug Administration ( F D A ) that checks the safety of food and the Environmental Protection Agency ( E P A ) that regulates the use of pesticides.

Although a great deal of attention is paid to food safety with regard to pesticide residues, there are also m a n y naturally occurring toxins c o m m o n l y found in food, some of which have been identified as potential carcinogens. The amounts of these carcinogens present in vegetables and fruit can range from 70 to 4,000,000 ppb (parts per billion U S , 10 ~ 9 ) , levels m u c h higher than the pesticide residues generally determined in food crops or animal tissues.1

Economic factors are also important in the use of pesticides. M o d e r n agricultural practices m a k e use of monocultures for the efficient production of crops. This provides concentrated areas of crop plants for which pests compete successfully as a result of their ability to quickly colonize and adapt. The high-yield cultivars and varieties developed and used in the Green Revolution proved to be somewhat less viable than the older varieties and required the input of pesticides. T h e resulting high crop yields and dramatic impact on the pests m a d e possible economic gains. Increased food production was a boon to developing nations trying to feed rapidly increasing populations, while in developed countries, the use of pesticides led to the development of more efficient food production. A n example of the economic dependence on pesticides can be demonstrated for Canada. If the phenoxy herbicides like 2 ,4 -D were banned, it is estimated that the potential loss to the Canadian economy would reach U S $ 4 1 0 million a year.

Concern for the global impact of economic development was addressed by M r s G r o Harlem Bruntland, the Prime Minister of N o r w a y , in a speech to the United Nations in 1987. In it, she called for the adoption of a policy of'sustainable development'. This policy recognizes the fact that economic development impacts on the environment and that consideration must be given to the future use of the earth's limited resources. In agriculture, this would involve the use of pesticides or alternative controls that are environmentally safe.

Pesticides: an overview

In the U S A , herbicides comprise about 70% of the pesticide market, while insecticides account for only about 25%.2 In contrast, the market in the Asia-Pacific region involves mainly insecticides. They comprise approximately 75% of the estimated pesticides used, with herbicides and fungicides representing 13% and 8% respectively of the market.3 Insecticides are used mainly for rice, cotton and vegetables, herbicides for rubber, oil palm, tea, coffee and cacao plantations, and fungicides for the control of diseases in tobacco, vegetables and bananas.

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Organochlorine compounds

Organochlorine compounds formed the first important class of synthetic pesticides, which includes D D T , heptachlor, aldrin, dieldrin, chlordane and endrin. Although D D T was first synthesized in 1874, it w a s not until 1937 that its insecticidal properties were recognized. The widespread use of organochlorine compounds , especially D D T , has played a significant role in minimizing the devastating effects of diseases in m a n y countries.

In general, organochlorine pesticides are easy to synthesize and inexpensive to manufacture. They also happen to be very stable and after m a n y years of their being used, residues were detected in the environment, especially in countries with temperate climates. T h e residues were found to have accumulated in wildlife, particularly in predatory animals and birds. National regulatory bodies placed restrictions on the use of organochlorine pesticides, cancelling registration for D D T in 1972, and restricting the use of endrin and other pesticides later on, in 1979. Continued, selective use was m a d e of these compounds by developing countries, primarily for vector control concerned with public health programmes for the control of malaria and trypanosomiasis. In 1984, an estimated 30,215 metric tons of D D T were used for this purpose.4

In addition to being persistent, organochlorine compounds , including non-pesticides like polychlorobiphenyls (PCBs) , are relatively volatile. In the tropics, this is the major cause of their disappearance, representing an estimated 90% of their loss.5 It also accounts for their global dispersion and entry into the aquatic ecosystem, with lakes and oceans being the ultimate environmental sink. T h e transport of chemicals in the environment, together with various m o d e s of degradation, are shown schematically in figure 1.

Figure 1.

The movement of chemicals within the environment.

ADSORPTION LEACHING RUN-OFF MICROBIAL DEGRADATION

29

R. Greenhalgh

O n e example of the global dispersion of organochlorine compounds is that of D D T residues in Japanese lakes, which are at as high a level today as they were before the moratorium on its use. Similarly, residues of chlordane derivatives and P C B s have been found in polar bear tissues.6 The latter report is disconcerting inasmuch as these animals form part of the diet of the native population and P C B residues have indeed also been detected in h u m a n breast milk. In the two cases, the organochlorine residues, both pesticide and industrial, are the result of farming and industrial operations taking place elsewhere in the northern hemisphere.

Organophosphorus compounds

The main insecticides used for agricultural purposes in 1964 were the organochlorine compounds. Following the moratorium on their use, they were replaced by organophosphorus and carbamate insecticides, so that by 1984, these two classes of insecticides represented an estimated 70% and 21% of the market respectively.7 The m o d e of action of both the organophosphorus and the carbamate compounds involves inhibition of the enzyme Cholinesterase. The insecticidal properties of the organophosphorus esters were first discovered in 1938, but were not exploited commercially until m u c h later when malathion, ethion, parathion, diaxinon, dimethoate, chlorpyrifos and fenitrothion became available. Although the organophosphorus compounds are still used extensively, problems associated with resistance in insects have developed, as they did in the case of the organochlorine compounds.

The replacement of the older, persistent organochlorine pesticides by the less persistent but more toxic organophosphorus and carbamate compounds is of concern in developing countries. More frequent applications led to a concurrent rise in hazards associated with their use, because of increased exposure. There is a need both for better training in the application of pesticides and for regulatory programmes for pesticides use in these countries.

Pyrethroids

These synthetic compounds are related to Pyrethrin I, the most potent of the six natural esters isolated from the chrysanthemum flower. They were developed in the 1970s and are a m o n g the most active insecticides k n o w n . 8 In the case of decamethrin, for example, synthetic modification of the active parts of the Pyrethrin I molecule has led to a 1000-fold increase in toxicity to houseflies. The pyrethroids are highly toxic to fish and aquatic invertebrates, and care must therefore be taken in their use close to bodies of water.

The development of the newer pesticides has dramatically reduced the pesticide load imposed on the environment, due to their greater activity. A comparison of the application rates for old and new pesticides on cereal crops is shown in Table 1.

Strategies for pest and disease control

The short breeding cycles of m a n y insects lead to the rapid development of resistance to insecticides, and this requires increased application rates or pesticides with new modes

30


Table 1.

Insecticides Organochlorine

DDT endrin

Herbicides Anilides

propanil

700 560

1000

Representative pesticide application rates for cereal crops (expressed as grammes active ingredient/hectare).

Organophosph

dimethoate malathion

Phenoxy-acids

2,4-D

orus

42(M80 850-1120

200-500

Pyrethroid

deltamethrin 6 Cypermethrin (a) 25

Sulphonyl ureas

metsulfuron-methyl 4-5

of action. Environmental concern also has promoted the search for more selective chemicals as well as more efficient ways of ensuring delivery of pesticides to target organisms. T o achieve the goals of increasing food production, environmental safety and sustainable agriculture, various strategies are n o w used for the control of insects and weeds, not all of which are chemical.

M a n y of the first-generation pesticides were the result of massive random screening programmes of both natural and synthetic chemicals. The search for novel approaches for disease and pest control led to the development of both biorational and biological procedures. Biological control involves the use of naturally occurring organisms to control and maintain pests below levels which are economically damaging. The biorational approach seeks to identify unique and vulnerable points in the life processes of the pest for exploitation and to develop environmentally acceptable control procedures. It m a y be considered as a m o r e comprehensive chemical concept of which the random screening of compounds is only a part. The testing for biorationals involves the use of in vitro biochemical systems rather than whole plants or insects.

Biorational approach

In the future pesticides will almost certainly continue to be used as the main strategy for insect and disease control, but within the context of an integrated control system. The development of biorational approaches using environmentally acceptable chemicals designed for specific purposes will steadily reduce reliance on broad-spectrum pesticides. The search for n e w chemicals requires further investigation of the living processes in insect and weed pests to identify unique target sites for potential control.

Insect control

Hormones Developmental endocrinology of insects is a favourable area for investigation since the growth and metamorphosis of insects are under the influence of hormones. The so-called juvenile hormone has been extensively investigated since, w h e n present above certain levels, it prevents development to the adult stage. Blocking the action of juvenile hormones early in the insect's life cycle could promote premature formation of undersized adults, whilst application late in the development of insects should prevent metamorphosis into the sexually-active adult.

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R. Greenhalgh

The insect moult is another interesting and characteristic process that warrants investigation. A 'primitive' enzyme is present in insects which has no k n o w n counterpart in higher organisms. It is present at high levels only during the moulting process and thus would be an ideal target site with potential for grasshopper and nematode control.9 Similarly, selected neurohormones and their inhibitors which have been identified in the insect nervous system m a y represent additional target sites.

Pheromones Insect communication is largely chemical in nature and has also served as a target for control procedures. Substantial research efforts are expended on the highly volatile communication chemicals produced by insects called pheromones. Behavioural control of insects through a variety of physical and chemical techniques has been tried. Pheromones, because of their unusual specificity and activity at extremely low concentrations, lend themselves to integrated control techniques.

The chemical messengers involved in reproduction are k n o w n as sex pheromones and have also been extensively investigated. M a n y insects release sex pheromones at specific stages in their life history, this release serving to ensure that the males find the females. If a synthetic sex pheromone is released simultaneously into the environment, it will interfere with normal mating by saturating the communication channels. Such chemicals m a y be used in traps to monitor the population levels of specific pests or to bring the pests into contact with chemosterilants or pesticides.

Secondary metabolites The search for environmentally safe compounds for controlling pests has led to the investigation of substances k n o w n as plant secondary metabolites, some of which are k n o w n to possess insect-repellant, insecticidal, antifeedant and anti-hormonal properties. These compounds, once isolated and identified, could also serve as models for synthesizing n e w and more active analogues for use in integrated control systems. Alternatively, they m a y be incorporated into other plants by genetic engineering techniques.

The neem tree, which is indigenous to India and B u r m a , is one source of insect feeding inhibitors and growth regulators. Field trials in India have shown that neem kernels are more potent for locust control than some insecticides. Azadirachtin is one c o m p o u n d isolated from neem seed oil which exhibits juvenile hormone behaviour as well as having pronounced behavioural and physiological effects. This compound is n o w in the process of being registered for use in developing countries. Again, synthetic modification of the molecule has enhanced the activity.

Plants can produce analogues of the hormones that are responsible for initiating the growth and developmental sequence in insects. S o m e plants produce analogues of juvenile hormones which act as controls over the moulting process. Thus two substances isolated from a commonly grown annual, Ageratum, were found to block juvenile hormone action, causing premature moulting of the larvae and death. These compounds, called precocenes, have been the subject of intensive research. Plants can also produce insect alarm pheromones which effectively protect it against predator attack. The hairs on the leaf of the wild potato, for example, when invaded by aphids release the same chemical that aphids release themselves when attacked by a predator. Thus the plant can repel a major pest by production of the pest's o w n alarm pheromone.

32


Microbial agents Fermentation of Streptomyces avermitilis produces several macrocyclic lactones that exhibit insecticidal and acaricidal activity, one being avermectrin B l a . This latter compound has been investigated for possible use in controlling different phytophagous pests of field and citrus orchard, as well as fire ants. Although poisonous to m a m m a l s , its exceptional toxicity to certain anthropods warrants the development of use patterns that will reduce its danger to non-target organisms.

Pathogen control

The environment contains m a n y pathogens—viral, bacterial and fungal— against which plants have to evolve natural defence mechanisms in order to flourish. Such mechanisms will be present in resistant varieties of plants. The infestation of wheat, corn (maize) and rice by the fungal genus Fusarium results in damaged crops as well as making the grain unusable as food or feeds due to the consequent mycotoxin contamination. Host-pathogen interaction and the development of resistant varieties of crops represent areas of great potential for non-pesticide control systems. Resistant varieties of plants are currently developed through Mendelian breeding, but in the future they are likely to be obtained using biotechnological techniques.

M a n y plant secondary metabolites have antifungal properties which m a y relate to their resistance to microbial pathogens. Plants also respond to cellular injury and pathogen attack by generating anti-microbial stress metabolites, called phytoalexins. These are part of the plant's defence mechanism which is associated with disease resistance. Investigations into the nature of resistance mechanisms and of those responsible for susceptibility have been examined in detail, but only separately. Information on h o w these two mechanisms interact is long overdue. The ability of molecular biologists to transfer genes from one plant to another also has tremendous potential for the manipulation of genes associated with resistance.

Biological control

While the potential for biological control is enormous, its use will be mostly within an integrated control system. For some time to come chemical pesticides will continue to play an important role in disease control using these systems.

The use of beneficial insects, i.e. parasites and predators, for the control of a pest has the advantage of being non-chemical. O n c e established, such operations are also long-lived. However, a detailed understanding is needed of the ecosystem into which the control agent is to be introduced, as well as of the population dynamics involved. Introducing a biological control agent from a foreign area m a y , in fact, create new problems since its o w n control agents m a y be absent in the n e w ecosystem. Indigenous biocontrol agents do not have this disadvantage, but their development still requires a knowledge of their life-cycle and mass-rearing techniques.

Insect control

The mass release of sterilized insects can result in increased competition for mates within a natural population and a significant reduction in the numbers of pest. Such a strategy has been successfully employed to manage screwworm flies in the

33

R. Greenhalgh

southwestern U S A and northern Mexico, involving an area of about 780,000 k m 2 . T h e infestation in livestock was found to have been reduced by 99%.10 The autocidal control of codling moth, Mexican fruit fly, boll weevil and tsetse fly has also been attempted. Evaluation of the technique in integrated pest management for the control of warble flies on cattle, however, has shown the technique to have limitations.

Several parasites and predators of the onion maggot have been isolated and three have been reared on a large scale.11 Investigations into their biology and ecology are being pursued to determine the environmental conditions required for their evaluation under field conditions within integrated control systems.

Since insect pathogens are usually non-toxic to vertebrates and plants, they hold out great promise as alternative pest control agents. Before registration of these products, however, the pathogen must be characterized with regard to strain, virulence, toxins produced, environmental persistence and dispersal mechanisms. In addition, its pathogenicity to vertebrates and plants must be k n o w n . The m o d e of action of the pathogen must be established, as well as transmission routes in order to optimize pathogen virulence. The identification of the toxins responsible for control and conditions favouring infection m a y allow their further manipulation.

Biological control by bacteria, fungi, viruses, nematodes, etc. is beset with difficulties which have prevented their widespread application. The bacterium Bacillus thuringiensis (BT) is a notable expection to this generalization.12 It has been used extensively in recent years as a commercial microbial insecticide in agriculture. The insecticidal properties of B T are attributed to a bipyrimidal shaped crystalline protein produced on sporulation and released on lysis of the cell. The toxin produced by B T is highly specific, with its toxic effects confined to certain butterflies, moths and flies. Improved strains are being developed which are toxic to a wider range of insects, including beetles. B T is environmentally safe and has also been used for the control of gypsy moth and spruce b u d w o r m infestations in forests.

Weed control

Although there are limitations to the use of fungi as microbial pesticides, such as their sensitivity to environmental conditions and fungicides, bioherbicides are available for weed control. The inoculum of plant pathogens is formulated and applied in a manner analogous to that of chemical herbicides. Bioherbicide programmes are designed to overcome or bypass restraints to disease development, such as low inoculum levels and weakly virulent pathogens, through the periodic application of the virulent inoculum onto a susceptible weed population. T w o bioherbicides are already on the market. Dev ine™, developed in 1981, consists of chlamydospores of Phytophthora citrophthora suspended in a liquid phase for the control of milkweed vine in citrus groves.13 The other, marketed under the n a m e of Collego™, consists of the dehydrated spores of Colletotrichum glaesporioides, and is used as a herbicide in rice and soya bean cultivation in the U S A . 1 4 Interest in bioherbicides has been revived in recent years.

Impact of biotechnology on biological control

Biotechnology has been associated with food since the times of early m a n , when naturally occurring micro-organisms were used in the making of bread, wine and

34


cheese. N o w , it is the ability of being able to manipulate micro-organisms by genetic engineering that is of interest, with the possibility of developing alternate pest control systems. Perhaps the greatest challenge to be met before the introduction of a wide range of microbial control agents will be to develop reliable, sensitive methods for assessing their potential impact on the environment and h u m a n health, such as already exist for chemical pesticides. It will also be necessary to determine the efficacy of each product in order to conduct risk-benefit evaluations.

Herbicide tolerance

It is ironic that current research is actively engaged in developing herbicide-tolerant varieties of crops such as canola (rape-seed). T h e first patent for an engineered crop gene, GlyphoTol, has been already issued in the U S A . The gene was obtained from Salmonella typhimurium and it endows tolerance to the herbicide glyphosate. However , incorporation of this gene into crops would permit a single treatment with the broad-spectrum, post-emergent herbicide glyphosate and eliminate the need for multiple sprays with more selective herbicides. This, in turn, would reduce the use of less environmentally safe compounds and also the total pesticide load o n the environment.

Insecticides

The B T gene has been genetically engineered into Pseudomonas fluorescans; this enables the latter to produce B T delta-endotoxin in soil, which can be used to control soil-borne pests around the roots of plants such as corn. Other possibilities include the transfer of the B T gene to other bacteria and viruses. Baculoviruses have a high potential for biocontrol, but a slow rate of kill makes them unsuitable for certain crops. Transferring the B T gene to baculoviruses would increase both the pathogenicity and rate of kill, while retaining host specificity. A s basic research continues to identify genes which control the formation of products responsible for natural immunity to pathogens or insects, further developments can be anticipated in which the release of the biocontrol agent into the environment is avoided.

Genetic engineering techniques for insects in general are not as advanced as for plants. S o m e progress can be expected in the development of autolethal techniques. This involves the introduction of reared insects carrying specific genetic characteristics that are lethal in the offspring. Successful use of this technique depends on the ability to breed the modified insects and introduce them into the environment in large enough numbers to enable them to compete with the naturally occurring strains during mating. This is an extension of the autocidal technique in which insects are sterilized by radiation or chemicals.

Summary

There is no one simple strategy by which m a n can control disease and pests such as insects or weeds. T h e use of chemicals is currently under attack because of their hazardous nature and effect on the environment. However, they will continue to be used as part of integrated pest management systems. The biorational approach should ensure that all new pesticides are designed to be specific in their m o d e of action, highly active and environmentally safe in accord with sustainable development.

35

R. Greenhalgh

Environmental effects and rapidly increasing pest resistance problems have helped to stimulate research into alternative pest control strategies. Several such systems are available that do not rely upon chemical pesticides, but these are long-term solutions and will require further research for their successful implementation as new control techniques.

Finally, progress in biotechnology will not only change the type of control agent used but also the amount of pesticide needed. A s with chemical pesticides, regulatory authorities will require extensive information on any genetically engineered organism used as a pesticide in order to assess its environmental impact and food safety as part of the on-going control on the use of pesticides. •

Bibliography

1. A M E S , B . N . (1989) Be most wary of nature's o w n pesticides, Los Angeles Times, 27 February. 2. A Y E R S , J. H . (1985) Status and outlook in the U . S . pesticide industry, American Chemical

Society (Abstract), Chicago, 9-12 September. 3. J O H N S O N , E . L . (1990) Pesticide regulation in developing countries of Asia and Pacific region.

In Regulations: a driving force in the evolution of Agrochemicals, eds: G . Marco, R . Hollingworth and J. Plimmer. A . C . S . Symposium series, in press.

4. E D W A R D S , C . A . (1986) Agrochemicals as environmental pollutants, pp. 1-20 in Control of pesticide application and residues in food: a guide and directory, eds: B . V o n Hofsten and G . Ekstrom. Swedish Scientific Press, Uppsala, Sweden.

5. K E A R N E Y , P . C , ISENSEE, A . R . and P L I M M E R , J. R . (1988) Contribution of agricultural pesticides to worldwide chemical distribution. In Toxic contamination in large lakes: sources, fate and controls of toxic contaminants, vol. Ill, ed: N . W . Schmidtke. Lewis Publishers Inc., Chelsea, U S A .

6. N O R S T R O M , R . J., S I M O N , M . , M U I R , D . C . G . and S C H W E I N B U R G , R . E . (1988) Organochlorine

contaminants in arctic marine food chains: identification, geographic distribution and temporal trends in polar bears. Environ. Sei. Tech., 22, 1063-1071.

7. S C H A U B , J. R . (1985) The economics of agricultural pesticide technology, pp. 15-26 in Agricultural chemicals of the future, ed: J. L . Hilton. Beltsville Symposia in Agricultural Research, vol. 8.

8. ELLIOT, M . (1977) Synthetic Pyrethroids, A C S Symposium Series N o . 42, Washington, U S A . 9. V A R D A N I S , A . (1985) A unique cyclic nucleotide dependent protein kinase, Biochem. Biophys.

Res. Comm., 125, 947-959. 10. K N I P L I N G , E . F . (1972) Sterilization and other genetic techniques. In Pest control: Strategies

for the future, National Academy of Sciences, Washington D C , U S A . 11. T O M L I N , A . D . , M I L L E R , J. J., H A R R I S , C . R . and T O L M A N , J. H . (1985) Arthropod parasitoids

and preditors of the onion maggot. J. Econ. Ent., 78, 975-981. 12. W E S T , A . W . (1984) Fate of the insecticidal proteinaceous parasporal crystal of Bacillus

thuringiensis in soil. Soil Biol. Biochem., 16, 357-360. 13. R I D I N G S , W . H . , M I T C H E L L , D . J., S C H O U L T E I S , C . L . and E L G H O L L , N . E . (1978) Biological

control of milkweed vine in Florida citrus groves with a pathotype of Phytophtora citrophthora. Proceedings of the IV International Symposium on Biological Control of Weeds, Gainesville, Florida, pp. 224-240.

14. T E M P E S T , D . O . and T E M P L E T O N , G . E . (1985) Mycoherbicides: progress in the biological control of weeds. Plant Dis., 69, 6-10.

36

Chemical drugs: new challenges

Camille-Georges Wermuth

The development of new chemical molecules for the treatment or cure of human and animal diseases goes through several stages in which chemistry plays a key role. The first stage is the identification and production of an original active principle; the second is the improvement of its performance in terms of potency, selectivity and safety; and the third is the adaptation of the molecule selected so that the doctor and the patient can make the best possible use of it.

The discovery of new active molecules has benefitted from progress m a d e in two different fields during the course of the last 20 years.

Firstly, there is the progress m a d e in instrumentation, especially increased sensitivity and miniaturization, the near-routine use of computer-aided design and image processing, and, above all, the growing use in pharmacology of molecular approaches. As a result, chemists n o w follow more rational procedures in the creation of new drugs, which even today are produced mainly by synthetic chemistry: of the 50 drugs sold most in the world, 48 are of synthetic origin. A breakdown by therapeutic category shows, somewhat surprisingly, that two of the first four are anti-ulcer drugs. These 50 drugs m a y be classified as follows: cardiovascular-14; anti-infective-13; gastrointestinal-4; for the central nervous system-3; for the musculoskeletal system-3; immune-response modulators 3; for the respiratory system-3; for the reproductive system-2; diagnostic-2; anticancer!; for the sensory organs-1; and hormones 1.

Secondly, m a n y drugs in future are likely to be the end-products of biotechnologies. Recent statistics show that of the products currently being developed there are three times as m a n y of biotechnological origin as in any other therapeutic category: 840 as compared with 278 anticancer, 276 antiviral and 237 anti-inflammatory synthetic drugs. The active products derived most frequently from biotechnologies are peptides and proteins.

Camille-Georges W e r m u t h is Professor of Organic Chemistry and Medicinal Chemistry in the Faculty of Pharmacy, Louis Pasteur University, Strasbourg, France. H e also heads the Molecular Pharmacochemistry Unit of the C N R S Neurochemistry Centre in Strasbourg. This unit is original in that it has three areas of responsibility: synthetic organic chemistry, pharmacology and graphic computer modelling. Research is essentially concentrated upon neuropsychiatrie pathologies; this has led to the development and synthesis of many research tools for the neurosciences and the development of a new psychotropic drug, minaprine, marketed in Europe since 1979 and currently being developed in Japan. Professor Wermuth has been President of the Medicinal Chemistry Section of the International Union of Pure and Applied Chemistry ( IUPAC) since 1988. His address: Centre de Neurochimie du C N R S , Département de Pharmacochimie Moléculaire, 5, rue Biaise Pascal, 67084 Strasbourg Cedex, France.


C.-G. Wermuth

Chemists then play a dual role in enhancing the usefulness of such products. They m a y establish and interpret structure-activity relationships, viz. determine a product's three-dimensional structure (by means of X-rays, N M R and conformational analysis) and study its interaction with the biological target (mainly through visual display on graphic computers), or else the product m a y be used as a model for making a simpler and chemically more stable synthetic analogue. Generally speaking, peptides and proteins of biotechnological origin cannot be taken orally, which accounts for research into substances that have simpler structures and mimic peptides but which are active when taken orally.

The role of chemistry in the manufacture of new drugs is therefore essential. It is doubtful, however, whether chemists have been properly trained to design and synthesize new drugs. It is therefore desirable for them to be trained in pharmaceutical chemistry. As a matter of fact, there is as m u c h difference between a basic chemist and a pharmaceutical chemist as between an architect and a naval architect. Just as an architect w h o wishes to become a 'naval' architect must have a basic knowledge of the sea and k n o w something about ships, a chemist w h o wishes to become a pharmaceutical chemist must k n o w something about biology (i.e. physiology, biochemistry, immunology and pharmacology) and about drugs. It would perhaps be useful in this regard to say what is meant by a drug and describe some specific aspects of its interaction with the h u m a n (or animal) body.

General features of drugs

W e shall n o w define what a drug is and then consider its specific characteristics, the ways in which it can be administered and its effect on the body.

What should a drug do?

Essentially, the purpose of a drug is to change a pathological situation (a state of disease) back into a normal physiological situation. It should be noted that molecules that make it possible to perform better than usual (psychostimulants, anabolic steroids, sexual stimulants, and so on) are not, for reasons of medical ethics, covered by the definition of drugs as perceived by the medical practitioner.

The second category of drugs are used to prevent disease or dysfunction. All vaccines obviously come into this category, although other preventive drugs, such as aspirin (against the risk of heart attack), antimalarial drugs, hormones (against osteoporosis), antioxidants, and vitamins and trace elements (against ageing), are also commonly used. Ideally, more and more preventive drugs should be made available, but in practice their development raises enormous clinical research problems.

The third group of drugs stands apart in that they are intended for people w h o are not ill. They include, for example, the contraceptive pill and a whole range of 'comfort drugs' such as tranquillizers, hypnotics, tonics and digestants.

Requirements peculiar to drugs

Drugs are expected to combine three qualities: specificity, efficacy and safety. In other words, the ideal situation would be as follows: there would be an appropriate drug for

38


each disease; it would be capable of treating, healing or preventing that disease; and its use would be entirely free from risk.

It is extremely difficult to meet these three requirements simultaneously, and this warrants ongoing research into new drugs. A n obstacle frequently encountered is the absence of an animal model for a given h u m a n disease or, where there is a model, its poor predictive value w h e n it comes to demonstrating therapeutic activity or to ensuring that there are n o toxic or side effects.

Lastly, there are still m a n y diseases that are not treated efficaciously; they include cancer, viral infections, especially A I D S , Alzheimer's-type senile dementia, arteritis, osteoporosis, atheroma, myopathies, arteriosclerosis, glomeruiitis and bilharziosis.

The three phases in the action of a drug

Basically, three main phases in the action of a drug can be identified. The first relates to the way in which it enters the body; this is the pharmaceutical phase. The second phase, which concerns its peregrinations and what happens to it between the site of entry and the site of action, is the pharmacokinetic phase. The third phase has to do with its actual effect once it reaches its target; this is the pharmacodynamic phase.

The pharmaceutical phase has to do with the means of administration (oral, percutaneous, intramuscular, rectal, etc.) and formulation (pill, capsule, injectable phial, cream, suppository, aerosol, etc.) of the drug. Adjustments can be m a d e to the physiochemical properties of the active principle (such as its solubility in water or lipids, its polarity and its acid or basic character) through slight changes in the chemical structure in order to optimize its solution in the biological fluids and its passage through the outermost membranes (for example the skin or the intestinal wall): in short, its penetration into the body.

In the pharmacokinetic phase all the factors enabling the drug to reach its site of action are studied.The site of action of a medicine is often separated in time and space from the site of administration or penetration. W e shall show later on in this article h o w certain structural modifications affect the fate of a drug within the body—the w a y it is absorbed, distributed, metabolized and excreted. Such modifications m a y be designed to affect either the distribution of the drug in the body or the duration of its action, although it is not always easy to dissociate the two things.

The pharmacodynamic phase is governed by the quality of the drug's interaction with its receptor site (the ligand-receptor interaction). It has long been k n o w n that most drugs act by attaching themselves to specific sites called receptor sites. T h e interaction between the drug and its receptor triggers off a series of physical, chemical and biological events. T h e overall effect induced by a drug is called the biological response. Most receptors are macromolecular, often proteinic, structures located either on the surface of membranes or in the cytoplasm. Biological responses to contact with the drug would, it seems, be the result of changes in the conformation of the receptor. These changes apparently cause enzymes to be released or modify ionic movements through the membranes by opening or closing pores in them. M a x i m u m effect is obtained w h e n there is perfect complementarity between the drug and the receptor; this result is approached through changes in the geometry or the distribution of electrical charges. The third phase therefore encompasses study of the ways in which the structure and the activity of drugs relate to each other. It is primarily in this third phase that the chemist

39

C.-G. Wermuth

will be active, but w e shall demonstrate that he or she plays an equally vital role in the other two phases.

From the idea to the molecule: the quest for active substances

Medicinal chemists have effective ways of optimizing the characteristics of an active substance. This m a y involve rather intuitive approaches such as the synthesis of analogues, isomers and isosteric molecules, the modification of cyclic systems, etc. or computer-aided design—especially the identification of pharmacophores through graphic modelling—or optimization by relating structure to activity in quantitative terms.

In any event, by taking up a lead, which could be a new chemical structure or a new mechanism of action, chemists can quickly develop more active and at the same time more selective and less toxic molecules. The real problem, however, lies precisely in the discovery or identification of an original line of research, since there is no laid-down formula for this important stage and no means of planning a laboratory's creative work. As a result, the discovery of n e w lead compounds is the most uncertain stage in the development programme of a drug.

U p to the 1970s such molecules were discovered primarily as a result of uncontrollable factors such as accidental observations, lucky finds, hearsay or the laborious screening of large numbers of molecules. M o r e rational approaches, based on knowledge of the structures or endogenous metabolites, enzymes and receptors, or on the nature of the biochemical disorders involved in disease, have since been introduced.

It m a y be noted from current analysis of the ways in which discoveries are m a d e that there are four rational approaches to the development of new active principles:

Chemical synthesis or extraction of thousands of original and patentable molecules without necessarily having any prior idea of their possible activity (screening).

Making improved copies of molecules that are already k n o w n and have been shown to be active. The aim of making such copies is to increase the molecules' therapeutic activity or reduce their side effects. A company m a y also wish to develop a drug similar to a successful one already on the market from a rival manufacturer; in such cases they have been referred to as ' M e too' drugs.

The selection of an existing model to work from—a natural molecule present in the body or a molecule whose effects on the body have been observed by chance. T h e synthesis of analogues, or products interacting with this molecule, then makes it possible to adjust its effect to secure beneficial curative or preventative action.

Lastly, the exploitation of a physiopathological hypothesis when knowledge of molecular pharmacology makes it possible to identify the mechanism that is out of order. O n e can then correct it by acting on different regulatory mechanisms such as enzymes, hormones and neurotransmitters.

Although all four approaches are still being followed, substantial progress has been m a d e in two of them, namely screening and rational research.

Screening is carried out more and more frequently through in vitro biochemical assays to measure affinity for a receptor by the displacement of tritiated ligands, or research to determine the enzymatic inhibition potency, for example. Other assays m a y concern research into antibiotic activity or evaluation of the effect of the given sample

40


on cell cultures or on isolated organs. All these approaches have three advantages in that they m a k e minimal use of laboratory animals, require only small amounts of the substance (a few milligrams is often enough) and lend themselves well to automation or standardized testing. Lastly, the samples studied are not necessarily well defined chemical molecules, and screening is increasingly being applied to plant extracts and fermentation products. These samples are first prepared in a roughly purified form by passage through a chormatographic column, and it is only when some activity of interest is observed that the active principle responsible for it is isolated and identified. Recent successes achieved using this approach have included the discovery of lovastatin, which forms the basis of a new generation of hypocholesterolemic agents that act by inhibiting hydroxymethylglutaryl-CoA reductase, and of asperlicin, as competitive non-peptide antagonist of cholecystokinin ( C C K ) used as a model for the development of simplified but extremely powerful synthetic analogues such as arylamides derived from 3-aminobenzodiazepinones.

Rational approaches are contingent on the progress achieved in molecular pharmacology and medical research. They have benefitted from the advent of new, highly sensitive research methods based on the use of radioactive elements, various visual display and imaging techniques and knowledge of the structure of enzymes and receptors gained from genetic engineering. The key element that will determine the rational approach to be taken will be identification of the physiopathological process involved in a given disease. It is on such a basis that the inhibitors of angiotensin convertase, currently the most widely used hypotensive agents in the world, were discovered.

The example of inhibitors of the conversion enzyme

The conversion enzyme catalyses two reactions that are thought to play a role in regulating blood pressure: (a) the conversion of angiotensin I, an inactive decapeptide, into angiotensin II, a powerful vasoconstrictive octapeptide; (b) inactivation of a nonapeptide, bradykinin, which is a powerful vasodilator. A n inhibitor of the conversion enzyme should therefore be a good 'candidate' for treating hypertension.

The first substance developed for this purpose was teprotide, a nonapeptide with a sequence identical to that of certain peptides isolated from a Brazilian viper. Teprotide is an effective inhibitor of the degradation of angiotensin I by the conversion enzyme. The presence of four prolyl residues and one pyroglutamyl residue makes this peptide relative resistant, but not sufficiently so to be administered orally:

pyro-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro-OH

Teprotide

In the search for a molecule with better bioavailability, researchers at the firm of Squibb based their reasoning on the analogy of the conversion enzyme to bovine carboxypeptidase A . Both enzymes are carboxypeptidases, but carboxypeptidase A removes only one amino acyl residue (C-terminal), whereas the conversion enzyme removes two. They also knew that the active site of carboxypeptidase A contains three elements for the interaction with the substrate (figure 1): an electrophilic centre forming an ionic bond with a carboxylic function, a site capable of forming an hydrogen bond with a C-terminal peptide bond, and a zinc atom solidly fixed to the enzyme and

41

C.-G. Wermuth

Substrate

R H o

Figure 1.

Models of the active site of carboxypeptidase A and of angiotensin-convertase. There are three types of bonding sites for carboxypeptidase A : ionic bond, hydrophobic interaction and zinc.coordination. Angiotensin-convertase brings the same interactions into play together with a hydrogen bond and an additional hydrophobic interactions. It will be noted that the transition from substrate to inhibitor is effected by replacing a scissile peptide bond by a break-resistant carbon-carbon bond.

Inhibitor

Substrate

O R, H O Inhibitor

General structure : ° C H ? n Y ^ o

R, H o

Example

forming a co-ordinate b o n d with the carboxyl group of the penultimate peptide b o n d (the one that will be broken).

O n the premise that the conversion e n z y m e functions in a similar w a y but simply one amino acyl residue further along (breaking the second instead of the first peptide b o n d , counting from the carboxyl terminal), the researchers at Squibb designed the m o d e l s h o w n in figure 1. According to this m o d e l , Af-succinyl alpha-amino acids could interact with each of the elements listed above through, respectively, their amino-acid

42


Table 1.

Comparative structures of some angiotensin-convertase inhibitors (the numbers indicated are mentioned in the text).

Substrate Formula

C02H

C02H

HO

H 3 C H

C02H

H CH3

C02H

HS

IC50(|iM)

330

22

1480

0-20

C02H

HS" X "O H 3 C H

C02H

0023 Captopril

Enalapril

43

C.-G. Wermuth

carboxyl (ionic bond), their amide linkage (hydrogen bond) and the carboxyl of the succinyl residue (co-ordination through zinc). These compounds could then act as competitive and specific inhibitors of the conversion enzyme.

A n Af-succinyl-amino acid series was then prepared, and N-succinyl-L-proline (1) (Table 1) did prove to be somewhat active (IC50 = 330 / iM) .* A m i n o acids other than L -proline give less active succinyl derivatives; this result is consistent with the fact that the terminal amino acid of several peptide inhibitors (especially teprotide) is also a prolyl residue in the C-terminal position. In this case in point, iV-succinyl-L-proline was the lead compound. It was then a matter of optimizing its activity. This was done by seeking a better interaction with the active site of the enzyme. T w o stages in this approach were decisive: the search for hydrophobic sites and the search for a better complexing agent for zinc (Table 1). Hydrophobic pockets were sought by substituting the succinyl residue with methyl groups (there were four possibilities, taking the regioisomers and stereoisomers into account). The alpha methylated structure (2) of the amide bond proved far more active than (1) ( I C 5 0 = 2 2 ¿ Í M ) . A high level of stereoselectivity was noted, the I C 5 0 of the antipode (3) of the c o m p o u n d (2) falling to 1,480/iM. Better zinc co-ordination was achieved by replacing the carboxyl residue with a thiol group. The improvement resulting from this modification was massive, as can be seen by comparing compounds (1) and (4) or (2) and (5). C o m p o u n d (5) with an I C 5 0 of 0-023/iM, is active w h e n administered orally and has been brought into therapeutic use under the n a m e Captopril.

It is interesting to note that the loss of affinity when the mercapto residue is replaced by a carboxyl residue m a y be offset by an additional hydrophobic interaction. Researchers at Merck, Sharp & D o h m e have developed Enalapril (6), which has a potency comparable to that of Captopril and in which additional hydrophobic interaction is brought about by a phenethyl residue.

Relating structure to activity in order to improve performance

A n active lead compound is rarely satisfactory as it stands; to increase its potency, eliminate side-effects or render its activity non-toxic the pharmaceutical chemist will need to synthesize a large number of analogues, guided in the work by proven methods such as the synthesis of isomers and isosteric molecules and the manipulation of cyclic systems or functional exchanges. Structure must be related to activity in order to correlate defined biological properties with the physiochemical or structural characteristics of the molecules.

In the so-called 'pattern recognition' approach, a data bank containing all the measured biological activities, including toxicity, of as m a n y compounds as possible is set up. Each molecule is then characterized by descriptors of physiochemical (molecular mass, density, partition coefficient, etc.) topological (atoms and bonds present, nature of the linkages, presence of cycles or of particular substructures), geometric (outline, volume and surface) and electronic (pKa, ionization potential,

* IC = Inhibition Concentration. I C 5 0 is the concentration expressed in gram molecules per litre capable of bringing about a 50% inhibition in the given enzyme.

44


electronic density, dipole m o m e n t , etc.) properties. A matrix calculation that is somewhat complex but which can be performed by computer makes it possible to select descriptor sets associated with a given biological or toxic activity and to channel syntheses along the desired lines.

The 'identification of pharmacophore pattern1 approach uses visual representations of molecules produced by interactive graphic computer programs. The graphics n o w available can produce a highly realistic three-dimensional representation of molecules. There is a large range of suitable software programs that give a very full description of the molecule under examination (geometry, conformational analysis, electronic environment, solvation and interaction capacity). Certain programs concentrate on the description of small molecules (less than 100 atoms), others on that of macromolecules (proteins, nucleic acids) or on crystallographic studies. Once stored in the computer's m e m o r y (in a low-energy conformation), bioactive molecules m a y be compared with each other or displayed in conjunction with simulations of their receptor sites.

If the three-dimensional structure of the receptor or target macromolecule is known—as is the case at present for m a n y enzymes—it is possible to set about the direct designing of a purpose-built active molecule. The fit between the ligand and its receptor is then optimized by establishing the best geometric and electronic complementarity possible between the two partners. The construction of the macromolecule is based on its structure as determined by X-ray crystallography or by N M R spectrometry. In parallel, the modeller builds the three-dimensional structure of the active—or presumed active—substance and can then manipulate the two molecules and thus observe directly the way in which the ligand adjusts at the active site. The experimenter m a y modify the geometry of the active molecule, change atoms and add or remove elements, monitoring the results of these modifications on the screen and retaining those that meet the most favourable conditions for an ideal fit. T h e molecules studied m a y be represented by different colours, the image m a y be enlarged or reduced, sections at different levels m a y be m a d e , and the experimenter can thus have a very clear representation of the interaction between the ligand and the macromolecule. A colour code m a y also be used to show up the electrostatic potentials present on the surface of the molecule and these m a y be adjusted to obtain the best possible electronic complementarity.

W h e n the three-dimensional structure of the target molecule is unknown, as is currently the case for most pharmacological receptors, indirect design is the only possible approach. The strategy used is k n o w n as the 'active analogue approach', in which a set of selective ligands of a given receptor or enzyme is compared in such a w a y as to reveal the molecular information they share in spite of seemingly very different chemical formulae. The object is therefore to determine the largest c o m m o n denominator of the set studied, in other words to identify the pattern responsible for the activity, i.e. the pharmacophore pattern.

Whether the approach taken is one of direct or indirect design, several advantages m a y be expected: (a) increased potency or selectivity within a chemical series; (b) the prediction of active substances possessing completely new structural characteristics; (c) the prediction of discriminating properties (agonists-antagonists, ligands of receptor subclasses); (d) better knowledge of the w a y in which ligands interact with their receptors; (e) an explanation of certain discrepancies or paradoxical pharmacological properties; and (f) the prediction of inactive compounds , which makes it possible to economize on the carrying out of syntheses.

45

C.-G. Wermuth

Control of pharmacokinetics

B y labelling active molecules with radioactive carbon (14C), it is possible to monitor the kinetics of their distribution in the body, the places where they are accumulated or stored and the routes by which they are excreted. O n e variant involving the use o f 1 1 C which has a very short half-life even makes it possible to apply this method to h u m a n beings.

Gas chromatography, coupled with mass spectrometry, makes it possible to measure infinitesimal quantities of active molecules in the different body components (brain, blood, urine, etc.) and to determine the structure of their metabolites. It often happens that the molecules in drugs are transformed, in the liver or brain for example, and that the metabolites thus found are more active than the parent molecules. Sometimes the metabolites have a different profile of action and m a y be the starting point for the development of a n e w drug.

Control over pharmacokinetic parameters and metabolic processes thus makes it possible to create molecules that m a y be distributed in the different parts of the body in a controlled w a y and will take a truly active form only after a biotransformational reaction. Prodrugs are a case in point.

Prodrugs, or emphasis on the form of administration rather than on the content

A n active substance administered orally m a y reach the target organ either with difficulty or not at all, a state of affairs described as poor bioavailability. The reasons for mediocre bioavailability m a y be poor absorption of the substance in the digestive tract, improper distribution of the substance in the various tissues or premature destruction through metabolism in the liver. T h e chemist will therefore be required to synthesize a new chemical derivative from the active principle, meeting the desired pharmacokinetic criteria, which, once it enters the living body, could regenerate the parent molecule from which it is derived. A n illustrative example of this procedure, which draws on the concept of the prodrug, is given below.

Antibiotics that are active when administered orally

M a n y major antibiotics are active when administered intravenously or intramuscularly, but not so when taken orally. This is rarely because they cannot withstand gastric juice acidity but more frequently because the body does not take up enough of them: their bioavailability is then said to be mediocre. T h e two parameters characterizing bioavailability are the fraction of the medicine administered which reaches the general bloodstream intact and the time it takes to d o so.

Ampicillin, a penicillin derivative, is a very widely used antibiotic because it combats a wide range of bacteria. Although this antibiotic is fairly well able to withstand the low p H (i.e. acid conditions) of the stomach, it is not very efficacious w h e n administered orally (two-thirds of the product administered is not taken up). In order to make ampicillin less polar and more lipophilic, a number of acyloxymethyl esters of this antibiotic, such as the derivative pivaloyloxymethyl or pivampicillin (figure 2) were prepared. Like most esters, pivampicillin is relatively stable in neutral solution, but it is quickly hydrolysed into ampicillin in the presence of esterases from the sera of various

46


O C H - C — N H

NHj Enzyme source Half-life (min.)

C O j H

Ampicillin

N o enzymes added Mouse serum, 1 % Rat serum, 1 % Whole h u m a n blood

103 <l <l

5

/"VoJ-\ = / I

NHj

N o n specific esterases

Pivampicillin

C H , I

C0 2 —CH 2 —OjC C — C H 3

CH3

CH,

C02 — CHj-OH * CH3 C—C0 2 H

CH,

Q—i-O

II C H - C NH

I NH; ^r—H

CC^H CH20

Figure 2. Pivampicillin results from blocking the acid function by a labile and lipophilic residue. In vivo, pivampicillin quickly releases ampicillin together with the pivalic acid and formol molecules. In vitro, the high hydrolytic activity of the esterases of rodents such as mice and rats is noteworthy. T h e addition of only one per cent of serum from these animals to an aqueous solution of pivampicillin with a p H of 7-4 and a temperature of 37CC reduces by more than 100 times the half-life of pivampicillin; the esterases contained in the sera of dogs or h u m a n beings are less active. Whole h u m a n blood, however, reduces the half-life 20 times.

47

C.-G. Wermuth

m a m m a l s . It is noteworthy that the hydrolytic activity of esterases from rodents such as mice and rats is very high. Adding only 1 per cent of serum from these animals to an aqueous solution of pivampicillin with a p H of 7-4 at a temperature of 37°C reduces by a factor of more than 100 the half-life of pivampicillin; the esterases contained in serum from dogs or h u m a n beings are less active, though whole h u m a n blood reduces its half-life by a factor of 20. All in vitro assays are instructive but they do not m a k e it possible to predict the real behaviour of pivampicillin throughout the body, and so they have been rounded off by studies in h u m a n beings. O n e group of patients were given an intramuscular injection of 250 milligrams of ampicillin and another were given an equivalent quantity of pivampicillin (385 milligrams, which is the dose capable of releasing 250 milligrams of ampicillin through hydrolysis) orally. It was observed that practically all the pivampicillin w a s absorbed and that 90 per cent of the drug present in the blood 15 minutes after administration was in the form of free ampicillin.

Pivampicillin is thus a good example of a prodrug, meeting four essential criteria: there is covalent bonding between drug and transport group; its o w n activity is minimal; there is rapid release in the body of the active drug and its transport group; and the latter is not toxic.

Chemical as opposed to galenical formulation

However powerful and selective it m a y be, an active molecule m a y have m a n y drawbacks for use in medical practice: it m a y have insufficient solubility, poor preservability or undesirable organoleptic properties (such as smell, taste or causticity); it m a y be painful to inject; etc. A suitably 'galenicaT preparation m a y correct these drawbacks. Substances administered orally m a y be given an enterosoluble coating if they are gastrosensitive; if they remain active for too short a period they m a y be prepared in a sustained-release formulation; or if oral administration is impracticable parenteral preparations m a y be considered.

The advantage of galenical preparation is that the active molecule is used as it is; in other words no chemical processing is required. Galenical preparation is not, however, a solution to all problems, and when it is inoperative chemical modifications of the active molecule must be considered, in which case the only remaining possibility is chemical formulation. The approach taken most frequently is to synthesize the simplest chemical derivative possible of the active principle to meet the desired pharmaceutical criteria. O n e of the major areas of chemical formulation is that of water-soluble drugs.

Water-soluble emergency drugs

The preparation of water-soluble forms of drugs is of vital interest since the water is the main solvent of living organisms. Water-soluble forms of active principles, even in pharmacological research, are desirable as they alone permit in vitro studies (of cell cultures, enzymatic systems, isolated organs and so on). For clinical purposes water-solubility is indispensable to the preparation of injectable drugs, especially for intravenous injection. It is also often of value for various topical applications such as eye, ear or nose drops, lotions, etc.

The typical route of administration of a drug is the intravenous one, and it m a y therefore be useful to mention its advantages and disadvantages. There are m a n y advantages: the drug goes straight into the bloodstream, which makes the method

48

CG. Wermuth

suitable for emergency treatment; it is a precise m o d e of administration, since the amount of the drug injected is strictly controlled; the injection m a y be spread out over a period of time (by the use of a drip) and stopped immediately if necessary; intravenous injection is the only possible route for products that are not absorbed or are destroyed in the digestive tract; substances which are irritating when injected subcutaneously or intramuscularly can very often be administered intravenously without difficulty (examples being alkaline and acid solutions and beta-chloroethylamine anti-cancer derivatives).

There are also some disadvantages: once injected, the drug cannot be removed, whereas when it is taken orally it is always possible to p u m p out the patient's stomach; if the drug is injected too quickly, it m a y cause cardiac failure owing to its very high local concentration in the heart (up to 400 times the desired final therapeutic concentration, since the total amount of drug injected into the vein enters the heart in one go); with certain drugs there is a risk of precipitation in the blood and therefore of embolism; lastly, injections carried out without proper sterilization m a y lead to bacterial contamination, which is particularly dangerous w h e n it occurs in the bloodstream itself.

The standard way of achieving solubility is to 'graft on' hydrophilic groups. This has, inter alia, allowed the development of corticoids for treating emergency cases.

Adrenocorticoids like cortisone have considerable anti-inflammatory and antiallergic properties but are almost insoluble in water. Yet in certain states of shock and serious allergic attacks (such as asthma, Quincke's oedema) emergency treatment is necessary. Water soluble derivatives have been prepared by grafting onto the alcohol - O H group in the 21 position of these steroids polar residues such as semi-succinate, phosphate, sulphate or metasulfobenzoate. Phosphates are chemically more stable and keep better than semi-succinates; they also give the highest blood concentrations w h e n injected intramuscularly or intravenously. Phosphate in the 21 position of betamethasone is one of the most active derivatives used in these emergency treatments (being 40 times more active than cortisone).

In this article w e have tried to illustrate the m a n y chemical facets involved in the production of drugs. While the discovery of n e w active principles is still the major challenge, it is equally true that the transformation of what is c o m m o n l y k n o w n as an active molecule into a genuine drug is a process with m a n y stages during which the work of the chemist is all-important. Taking the pharmacodynamic, pharmacokinetic and pharmaceutical phases into consideration thus provides daily challenges for the synthetic chemist. However, even when the active substances have been produced by biotechnology, the chemist is again called o n both to throw light on their often complex structures and to develop simpler but chemically more stable synthetic analogues, if possible also at a lower cost price. •

To delve more deeply

W E R M U T H , C . G . (1984) Designing prodrugs and bioprecursors, pp. 47-72 in Drug design: fact or fantasy? Academic Press, London.

S N E A D E R , W . (1986) Drug discovery: the evolution of modern medicines. John Wiley, N e w York.

49

Strong fibres from flexible chain polymers

Miroslav Raab

During the last two decades polymer physicists have developed techniques for effective control of the supermolecular structure of semicrystalline polymers, bringing about a dramatic change in their mechanical characteristics. High-performance and lightweight polyethylene fibres are now superior to classical materials such as steel, especially as far as strength vs. density is concerned.

High-performance fibres from flexible chain polymers represent a new class of materials. Having originated from fundamental academic research in polymer physics, they are n o w attracting the interest of large industrial companies and have a significant potential impact on our everyday lives. Efforts to convert cheap commodity plastics into valuable specialized products have been particularly successful in the case of linear polyethylene. Extremely strong and lightweight polyethylene fibres n o w to find applications ranging from marine ropes and bulletproof jackets to composite materials. This article gives a short survey of the various possible routes that lead from common-grade materials to extremely strong products just by changing physically the structural order of their molecules.

Morphology of semicrystalline polymers

Polyethylenes, polyamides and other semicrystalline polymers, w h e n crystallized from solution, form crystalline lamellae with molecular chains more or less regularly folded and aligned perpendicularly to the largest surface plane. (The crystal regularity depends on molecular structure and the conditions of crystallization.) Basically the same lamallae are also present in a polymer solidified from melt, but in this case they are embedded in a matrix of amorphous polymer. However, narrow temperature 'windows' exist for m a n y semicrystalline polymers, where the formation of extended-chain crystallites is also possible. Moreover, the external pressure and the existence of a

Miroslav Raab is Senior Scientist at the Institute of Macromolecular Chemistry of the Czechoslovak Academy of Sciences in Prague, Czechoslovakia. A graduate of the University of Brno, he received his Doctoral degree for the Charles University in Prague in 1968. His professional interests include structural explanation of the mechanical behaviour of polymeric materials, but he is also active in the popularization of materials science through Czechoslovakian journals, radio and television. His address: Institute of Macromolecular Chemistry, Heyrovsky Sq. 2, 162 06 Prague 6, Czechoslovakia.


M. Raab

flow field generally support crystallization of extended-chain crystals. In particular, so-called 'shish-kebabs' can be thus produced by stirring a slightly supercooled polymer solution. These crystalline formations consist of a central fibrillar extended-chain core ('shish') to which folded-chain lamellae ('kebabs') are attached perpendicularly.

Macroscopically isotropic semicrystalline polymers with lamellar morphology are relatively soft and weak materials, but the strength of the C - C bond in the polymer backbone is still very strong, similarly to that of diamond. The macroscopical strength and rigidity can be therefore increased markedly by orientation of molecular chains, particularly in the case of linear polyethylene.

Orientation and chain extension

The original meaning of the verb 'to orient' was to face or point towards the east ('on'ens' in Latin), specifically to build churches with the main altar at the eastern end. However, since the second half of the last century, orientation has also come to m e a n an arrangement or alignment of atoms or molecules along certain preferential directions. The classical assessment of orientation was developed for inorganic crystals and is based on the birefringence (i.e. the difference in refractive indexes) of visible light. Later, the same method was adopted to measure the orientation of natural and synthetic fibres. Herman's Orientation Factor is defined as:

fH = A n / A n 0 ,

where A n is the difference in refractive indexes along and across a fibre and A n 0 is the corresponding difference along and across the molecular chain. Obviously fH = 0 for an isotropic material; fH = l and fH= —0-5 for a perfectly oriented polymer along and across the orientation direction, respectively.

Soon after the preparation of the first fully synthetic fibre (nylon) by Carothers in 1935, it was recognized that the stiffness and strength of m a n - m a d e fibres could be substantially enhanced by cold drawing. Originally this was ascribed to molecular orientation. M o r e recently, however, attention has been paid to polymer chain extension which is also induced by deformation and which for some mechanical properties becomes even more important. T w o molecules shown in figure 1 have the same orientation factor, but differ substantially in their conformation. In particular, chain extension plays a key role in determining the macroscopic tensile strength.

Small-angle neutron scattering is an important tool in the evaluation of polymer chain extension. The method is based on the fact that neutron scattering changes markedly when hydrogen atoms in polymer chains are replaced by deuterium. Unfortunately, this technique fails in the case of polyethylene, since deuterated and c o m m o n (protonated) polyethylenes are incompatible, so that the deuterated polymer tends to form segregated domains. However, molecular deformation m a y also be assessed from shrinkage of a drawn specimen on rapid heating.

The riddle of cold drawing

In semicrystalline polymers plastic deformation can either be homogeneous along the whole specimen or localized in a 'telescopic' neck. The material which has passed the

52


Figure 1.

T w o molecules showing the same orientation factor but differing in molecular extension (from G a o , Mackley and Nicholson, 1990).

neck carries the same load as the non-oriented portion of the sample, in spite of the drastically reduced cross-section. This is possible because the drawn polymer exhibits both high modulus* and tensile strength. Microscopical investigations and X-ray diffraction show that the semicrystalline polymer undergoes distinct structural changes upon drawing. The original lamellar morphology is transformed into a fibrillar structure with molecules oriented preferentially along the direction of draw. The mechanism of the transformation is, however, still an object of controversy.

The classical model supposes that the transformation from lamellae to microfibrils merely demands a chain tilt and slip in a limited region but is not seriously affected by what happens at a distance. M o r e recently, an alternative concept has been proposed in which the transformation from the lamellar to fibrillar morphology depends solely on phase transition. The stored mechanical energy is supposed to cause local 'melting' of the semicrystalline polymer at the draw temperature. In the second step the action of mechanical stress causes a rapid extension and strain-induced recrystallization of the amorphous 'melt'. It is supposed that the resulting morphology of the cold-drawn polymer consists of 'shish-kebabs' similar to those produced by crystallization from polymer solution in a flow field. The high strength and rigidity of the oriented polymer is then ascribed to covalent bonds in the extended chain core. The proportion of these load-bearing molecules can, however, vary dramatically according to the drawing conditions.

T w o routes to extremely strong and rigid polyethylene

The preparation of strong extended-chain structures from flexible-chain polymers was particularly successful in the case of polyethylene. The polyethylene chain has no side groups and the smallest cross-sectional area per chain. A tightly packed array of parallel polyethylene chains has therefore a high density of load-bearing elements and should be capable of constituting a very strong material. Estimates of the theoretical strength of oriented polyethylene along the direction of the chain are between 16 and 36 G P a and calculations of the maximal attainable modulus of elasticity vary from 180 to 340 G P a . (The experimentally observed tensile strength and modulus values of

* Modulus, a measure of elasticity: force needed to double the length of a rod of unit cross-section of a given material on which it were applied (Young modulus—Thomas Young 1773-1829).

53

M. Raab

c o m m o n high-density polyethylene are only 003 G P a and 2 G P a , respectively.) Actual oriented systems inevitably contain a number of flaws and molecular irregularities (such as kinks, chain ends, molecular twists) and theoretical values can never be attained in practice. Nevertheless, the development of techniques to bring about a more complete orientation and extension of macromolecules has resulted in fibres with surprisingly good mechanical characteristics.

T w o general routes are used to achieve high-modulus polyethylene (see table 1). T h e most straightforward way uses the deformation of neat polyethylene (usually of med ium molecular weight) in the solid state or in the melt. The major factor here is the establishment of a very high degree of plastic deformation or elongational flow. This m a y be obtained by special procedures of drawing at a temperature above the secondary transition of the crystalline phase (about 90°C), but still well below the polymer melting point. At this temperature range polyethylene shows a general relationship between the modulus and the draw ratio. The molecular weight distribution and initial morphology are equally important. The process leads to a high modulus, but relatively low strength and high creep products. (The Celanese C o m p a n y has announced the production of this type of fibre.) Similar results can also be obtained by solid-state extrusion: conventional ram extrusion, hydrostatic extrusion, extrusion combined with drawing and the so-called solid-state coextrusion. A split billet is used in the latter case where two external parts form a sheath and the internal plate between them is extruded. B y this technique one of the highest strength values has been obtained by solid state deformation for a polyethylene sample with the molecular weight ( M w ) of 2 0 x 10"6g/mol. Another solid-state technique is rolltrusion. In this case the workpiece is highly compressed between thermostatic rollers and simultaneously drawn. Recently, it has been shown that the orientation of linear polyethylene at a temperature close to its melting point but already in the softened state can also produce materials with high modulus and strength.

The second possible route uses ultra-high molecular weight linear polyethylene ( P E - U H M W ) with a suitable solvent, i.e. it involves solution- or gel-spinning. In 1976 Zwijnenburg and Pennings developed a technique for continuous longitudinal growth

Table 1.

Basic routes to high-performance polyethylene and properties of products.

Route

Solid-state- or melt Solid-state

drawing Solid-state

extrusion Co-extrusion Rolltrusion Melt-drawing

Molar mass (10-6g/mol)

-drawing

01

O05 20 O06 095

Orientation in solution Crystallization

in flow field Suspension-

spinning Gel-spinning

>10

>10 >10

Young modulus

(GPa)

90

6 220 40

60-80

120

120 200

Tensile strength (GPa)

04

05 20 07 1-5

4-7

3-8 6-2

Breaking strain (%)

—

— 1 5

—

5

5 —

D r a w ratio

30

35 250 25

60-80

5

100 300

54


Figure 2.

Schematic representation of the gel-spinning process (from Lemstra and Kirschbaum, 1985).

•

Solution

-ob-

\

r Extrusion

)

• b " - r i

Water bath

I i)L

Drawing

V 2 > V ,

1 J0

of fibrillar crystals of linear polyethylene subject to Couette flow. The Couette apparatus is basically a rotational viscometer comprising two concentric cylinders, where the inner cylinder is rotating at controllable speed in a polymer solution. A seed crystal is inserted into the gap between the cylinders and w h e n its tip contacts the moving surface, it starts growing. A take-up roll is then switched on and a continuous fibre is slowly produced. With a 0-5% solution of polyethylene Hostalen G U R ( M w = 1-5 x 106) and a temperature slightly above the crystallization point (135°C) an extended chain polyethylene fibre was obtained. U p o n subsequent drawing moduli of 1 2 0 G P a and tensile strength values of 5 G P a can be reached.

The gel-spinning technique developed by Smith and Lemstra produces fibres of comparable strength, but at a substantially higher production rate (figure 2). In this process ultra-high molecular weight polyethylene is dissolved in decalin at a high temperature. The viscous solution is then spun into a water bath and the resulting 'gel fibres' are subsequently drawn at higher temperature. T h e solvent is removed simultaneously and possibly recycled. T h e so-called suspension technique which uses paraffin oil as solvent is a similar procedure. These techniques have been commercialized by the Allied Corporation in the U S A (as Spectra fibres), D S M in the Netherlands (Dyneema) and Mitsui in Japan (Tekmilon). Thus, it can be seen that elaborated polymer physics has enabled us to convert cheap high-volume polymers into valuable high-performance materials (see figure 3).

High-performance nylons and some other polymers

The conventional drawing of polyamide 6 and polyamide 66 results in a m a x i m u m draw ratio of about 5, m u c h less than in the case of polyethylene. C o m m o n nylon fibres have typical moduli of 4 -5 G P a and tensile strength values of about 0-5 G P a . O n the

55

M . Raab

Figure 3. Performance and price

Schematic relation I between production T Specialities volume, performance I and price of polymeric 1 materials (from Lemstra I and Kirschbaum, 1985). I

Structural polymers

Modified polymers

High-volume polymers

Volume

other hand, the theoretical modulus of polyamides ranges from 180 to 300 G P a and the theoretical strength should am ount to about one-tenth of the tensile modulus. Hydrogen bonds in both crystalline and amorphous regions hinder chain sliding, thus preventing the drawing and orientation to a higher degree. Progress in the ultra-drawing of polyamide 6 and other aliphatic polyamides, however, is a challenge. The high melting point of this polymer (225°C), together with the high modulus, might lead to a very promising material in the future.

Partial success in this direction has already been achieved by the use of several procedures based on the weakening of hydrogen bonds. Higher draw ratios can be thus achieved. O n e method is the plasticization of polyamide 6 with liquid a m m o n i a which is subsequently removed after solid-state coextrusion. Such plasticization occurs only in the amorphous phase, but results in a modulus increase up to 13 G P a . Another method uses iodine as a plasticizer, which penetrates both amorphous and crystalline phases. Immersing a polyamide 6 film into a potassium iodide solution causes a complete structural transformation and a dramatic change in the drawing behaviour. After drawing, the absorbed iodine can again be removed by washing with sodium thiosulphate solution.

Dry-spinning of polyamide 6 from a formic acid/chloroform consolvent mixture followed by hot-drawing has produced fibres with moduli up to 12 G P a and tensile strength values up to 11 G P a . Another process of producing high-modulus and high-strength polyamides involves melt-spinning and drawing of mixtures of polyamide 6 and small amounts of lithium salts. A modulus of 13 G P a was thus obtained. Finally, zone-drawing and zone-annealing methods are promising from the technological point of view. Both processes rely upon a narrow band heater which moves along a stressed

56


fibre. The temperatures are 80°C for the drawing zone and about 180°C for the annealing zone. The best results are obtained if zone drawing is followed by multi-step annealing.

Techniques for the production of high-performance oriented fibres have also been investigated for other flexible-chain polymers. Thus , an ultra-oriented polyoxymethylene fibre, which has a Y o u n g modulus of 6 0 G P a , was produced by drawing under dielectric heating. Fibres of ultra-high molecular weight polypropylene prepared by the gel-spinning method at a draw ratio of 100 attained a modulus of 4 0 G P a and a tensile strength of l-6GPa.

Structural models

Several models have been proposed for the structure of highly oriented semicrystalline polymers with enhanced mechanical properties. They differ in detail, but share some c o m m o n features. Namely, they all suppose the existence of fully extended and straightened polymer chains interconnected s o m e h o w to form a continuous crystalline phase at the molecular level. They are termed 'continuous crystals', 'crystalline bridges' or 'tie molecules' in the various models. In all models the high mechanical properties are ascribed to this connected phase which provides for an efficient transmission of axial force.

For example, the continuous crystal model developed by Porter (and also m u c h earlier by Staudinger) assumes the existence of a parallel array of straightened chains where the defects (chain ends) are randomly distributed. O n the contrary, the Fischer model involves the local concentration of defects, giving rise to density fluctuations.

It is difficult to distinguish experimentally between the various models, but a comparison of experimental mechanical data and the calculated theoretical values seem to support the concept of the continuous crystal. The observed superheating of melting along with morphological observations also provide evidence of high crystal continuity.

Applications

Only high-strength polyethylene has been so far used on a commercial scale. Its most notable feature is the extraordinarily low density, about two-thirds that of Kevlar and half that of carbon fibres. Consequently, extremely strong polyethylene has markedly higher specific strength than any other strong reinforcing fibre currently used in composites (figure 4). High-performance polyethylene has found successful applications in sail cloth, fishing nets and marine ropes. It has been the unique combination of physical properties which has led to the relatively rapid acceptance of the new material in these areas. Particularly favourable properties are the low weight, low friction, low swelling in water and good weather stability. Because of their inherent ductility, polyethylene ropes have a reportedly better resistance to dynamic fatigue and mechanical shock then Kevlar.

In some composite applications the low adhesion to c o m m o n matrices, low compression strength and low melting temperature could play a limiting role. Polyethyelene fibres also have a higher extensibility than other reinforcing fibres. This,

57


Figure 4.

Stress-strain traces of rigid and strong fibres intended for use in composites. Stress in related to linear density (1 tex = l(r6kg/m) (from Lemstra and Kirschbaum, 1985).

Stress (N/tex)

3

2

1

Carbon . fibre / u t

1 1 1

/ A \ \ \ \ \ High-performance / \ \ \ \ \ \ v \ polyethylene _

X U ^ — Aramids

Glass fibre -

JteeUvire ^ P d y a m i d e s

1 1 1

5 10 15

Breaking strain (%)

however, could be of advantage in applications where toughness and high energy absorption are desirable. Moreover, polyethylene and carbon fibres can be combined in interesting hybrid composites.

Perfectly oriented polyethylene fibres can be rapidly overheated to at least 20°C above the usual polyethylene melting temperature. This makes possible their use as reinforcing fibres, not only in polyester and epoxy matrices, but also in thermoplastics, including polyethylene itself. T h e resulting one-polymer two-phase composites reinforced with strong and ductile fibres effectively block crack-propagation in a similar way to s o m e biological tissues in the natural world, for example the leaves of the broad-leaved plantain {Plantago major). Moreover, they could be easily recycled. •

Acknowledgement

I a m indebted to D r Milos Hoff, I M C Prague, for his valuable c o m m e n t s on this article.

Bibliography

G A O , P., M A C K L E Y , M . R. , and N I C H O L S O N , T. M . (1990). Development of anisotropy in ultrahigh molecular weight polyethylene, Polymer, 31, 237-242.

L E M S T R A , P. J., and K I R S C H B A U M , R . (1985). Speciality products based on commodity polymers, Polymer, 26, 1372-1384.

L I M , J. G . , G U P T A , B. S., and G E O R G E , W . (1989). The potential for high-performance fibre from nylon 6, Prog. Polym. Sei., 14, 763-809.

W A R D , I. M . (ed.) (1975). Structure and Properties of Oriented Polymers, Applied Science Publishers, Barking, U K .

W E E D O N , G . C , and T A M , T . Y . (1985). Properties and applications of extended chain polyetheylene, in Symposium 'High Performance Fibres, Textiles and Composites', Manchester, U K .

58

C F C s and their alternatives

Michael Freemantle

Chlorofluorocarbons (CFCs) are used as refrigerants, aerosol propellants,foam blowing agents and solvents for cleaning. However, they deplete ozone in the stratosphere and contribute to the greenhouse effect. The chemical industry is now developing a range of alternatives to CFCs.

In April 1930, the U S trade journal Ice and Refrigeration reported the discovery of a new refrigerant which, it predicted, would bring about a vast improvement in the world's environment. According to the journal, the u n n a m e d refrigerant was 'nontoxic, non-flammable' and had 'very desirable engineering characteristics for refrigeration'.

This new environmentally friendly c o m p o u n d was conceived as a 'miraculous refrigerant' because of its desirable properties. T h o m a s Midgley and Albert Henne, two chemists at the Frigidair Division of General Motors in the U S A , developed the new refrigerant to replace toxic and inflammable ones such as sulphur dioxide, methyl chloride, ethyl chloride and propane.

The new refrigerant was dichlorodifluoromethane. It was given the n a m e F R E O N -12. Joint production by General Motors and D u Pont began in 1931 using a simple one-step process involving the reaction between tetrachloromethane and hydrogen fluoride catalysed by antimony (V) chloride:

C C 1 4 + H 2 F 2 U C C 1 2 F 2 + 2HC1

The new compound was considered a great development. Not only was it nontoxic, non-flammable and therefore safe, but it was also stable, non-volatile and non-corrosive. It therefore replaced carbon dioxide and a m m o n i a in m a n y applications.

Michael Freemantle is the Information Officer for the International Union of Pure and Applied Chemistry (IUPAC) in Oxford. H e is responsible for the I U P A C affiliate membership programme and edits the I U P A C news magazine Chemistry International.

Dr Freemantle was Lecturer in Physical Chemistry at the Polytechnic of the South Bank, London and Associate Professor of Physical Chemistry at the University of Jordan, A m m a n . H e has worked as an editorial consultant in science education for Unesco and written eight books on chemistry and science, including Chemistry in Action published by Macmillan Education in 1987. H e may be contacted at the I U P A C Secretariat, Bank Court Chambers, 2-3 Pound W a y , Templars Square, Cowley, Oxford O X 4 3YF, UK.


M . Freemantle

The C F C family

In the 1940s D u Pont developed a range of fluorocarbon compounds to satisfy new demands from industry and society. These C F C s , as they became known, were inexpensive, efficient and easy to use. They increased the market for refrigeration and air conditioning.

In the following decade, C F C s began to be used as blowing agents for plastic foams, in fast-food packaging for example, and as solvents, especially in the dry-cleaning industry. The 1950s also saw the start of the use of C F C s as aerosol propellants. In this type of application C F C s were released into the atmosphere—unlike refrigerants which were confined to a closed system.

A wide variety of other applications were also found for C F C s , for example in sterilizing surgical instruments, medical oral inhalation products, and degreasing and cleaning electronic components.

In the early 1970s about one million tonnes of C F C s were being produced each year, with C F C - 1 1 and C F C - 1 2 each accounting for about 300,000 tonnes per year. By 1988 world production was just over 11 million tonnes per year.

The pattern of usage varies widely throughout the world. For example, in the U K the primary market for C F C s is as aerosol propellants (62% of the market). Two-thirds of the 600 million spray cans used each year contain C F C s . F o a m blowing agents account for 18% of the market, solvents 12% and refrigerants 8%. In the U S A , on the other hand, refrigeration and air-conditioning accounts for 35% of the market. F o a m blowing also accounts for 35%, followed by solvents (18%) and sterilants (6-5%). The non-essential use of C F C s as aerosol propellants has been prohibited in the U S A since 1978, the prohibition eliminating 95% of the aerosol market in the country.

Table 1 shows the five major commercial C F C s and their main uses. The most important factor in the selection of a C F C for a particular application is boiling point. The C F C s listed in the table have boiling points ranging from 48°C (for CFC-113) to -39°C(CFC-115) .

CFCs-11 and -12 are methane-based compounds whereas the others are based on ethane. The table also includes three related compounds known as halons. These halogenated methane and ethane compounds contain bromine, and are principally used in fire extinguishers.

Table 1. CFCs, halons and their major uses.

Compound Formula Uses

Methane-based CFCs: CFC-11 C C 1 3 F Aerosols, refrigeration, air-conditioning, cleaning, foams C F C - 1 2 C C 1 2 F 2 Aerosols, foams, refrigeration, air-conditioning, sterilization

Ethane-based CFCs: CFC-113 C C 1 2 F C C 1 F 2 Refrigeration, cleaning, foams CFC-114 C C 1 F 2 C C 1 F 2 Aerosols, refrigeration, foams CFC-115 C C 1 F 2 C F 3 Refrigeration, air-conditioning, foams

Halons: Halon-1211 CF 2BrCl Portable fire extinguishers Halon-1301 C F 3 B r Total flood fire-extinguishing systems Halon-2402 C2F,,.Br2 Fire extinguishers

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CFCs and their alternatives

The numbering system used to label C F C s was devised by scientists at D u Pont in the early days of C F C development and production. The first digit on the right is the number of fluorine atoms in the molecule. The second digit from the right is the number of hydrogen atoms plus one. The third digit from the right is the number of carbon atoms minus one. (If this is zero it is omitted.) For example, C F C - 1 1 5 has five fluorine atoms (first digit on right); no hydrogen atoms (second from right: 0 + 1 = 1); and two carbon atoms (third from right: 2 — 1 = 1). The letters 'a' or ' b \ used for some C F C alternatives (see below), designate an isomeric form of a compound.

Ozone depletion in the stratosphere

Ozone was detected in the stratosphere in 1881. It is formed from the interaction of oxygen in the troposphere with sunlight. In 1930, Sidney C h a p m a n explained the mechanism for its photochemical formation in terms of the following reactions:

0 2 + hv -> 20- at wavelengths below 230 n m

O + 0 2 - > 0 3 ozone

0 3 + hv -• 0 2 + O at wavelengths 200-290 n m

These reactions are most pronounced above the equator and the tropics because of the strong solar radiation in these regions. Winds in the stratosphere carry the ozone around the earth towards the polar regions.

Questions about depletion of stratospheric ozone were first asked in the early 1970s. In 1971, there were suggestions that the nitrogen oxides emitted as combustion products in the exhaust gases from the engines of supersonic aircraft flying in the stratosphere might effect ozone levels.

In 1974, two American scientists, Rowland and Molina, linked the destruction of ozone in the stratosphere to C F C s . They claimed that chlorine radicals catalytically decompose stratospheric ozone and that the main anthropogenic source of chlorine in the stratosphere could be C F C s . They estimated that if the rate of production of C F C s were to continue, half a million tonnes of chlorine would accumulate in the stratosphere each year. This, they suggested, would double the natural rate of ozone decomposition and, as a result, ozone would be depleted by 7 to 13%.

In 1985, J. C . Farman of the British Antarctic Survey reported a seasonal reduction of ozone over Halley Bay. Measurements in 1987 showed that 95% of the ozone between altitudes 14 and 23 k m was destroyed in less than two months. The total ozone column was reduced to 40% of its pre-1979 thickness. These findings were confirmed by the Ozone Trends Panel (OTP) set up in October 1986 by the U S National Aeronautics and Space Administration ( N A S A ) and other agencies. The committee, which consisted of an international group of atmospheric scientists, re-examined data from satellites and ground stations.

In 1987, O T P reported that atmospheric ozone levels at middle and low latitudes decreased by 2-5 per cent between 1978 and 1985. At high northern latitudes, between 50° and 60°, ozone concentrations fell 6% compared with the level in 1970. O T P attributed part of this depletion to C F C s . The drop in the mean total ozone concentration over Halley Bay for Octobers from 1957 to 1985 closely matched the build-up of C F C s in the southern hemisphere.

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M . Freemantle

Chlorine radicals (Cl) are produced by photolytic dissociation of C F C s in the lower stratosphere. The following reactions for CFCs -11 and -12 respectively are typical:

CCl3F + hv-CCl2F+Cl-

CCl2F2 + hv-CClF2+Cl-

These radicals then become available to catalytically destroy ozone at all altitudes in the stratosphere in a complex series of processes including the following which involve chlorine monoxide (CIO):

Cl+0 3 -C10+0 2

C10+0->Cl+02

Scientists estimate that a single chlorine radical can react with up to 100 ozone molecules. Moreover, they predict that C F C s , because of their stability, can survive in the stratosphere for up to 100 years.

Halons are even more destructive. They release bromine radicals (Br) which form bromine monoxide (BrO) in similar reactions to those shown above. The concentration of halon 1301 in the stratosphere has been increasing at the rate of 5% per year.

The total stratospheric concentration of chlorine today is about 3 parts per billion (ppb, parts per 109 molecules of air). This compares with 0-6 ppb a century ago and 2ppb in the late 1970s. The chlorine is mainly in the form of C F C s and their photochemical by-products such as C I O . Bromine, in the form of halons and bromine monoxide, is also present at about 002ppb .

Recently, there has been some concern that halogenated anaesthetics such as halothane (CF 3 CClBrH) , enflurane ( C F 2 H O C F 2 C F C l H ) and isoflurane ( C F 2 H O C H C l C F 3 ) m a y also contribute to stratospheric ozone depletion. However, research by A . C . Brown has shown that anaesthetics are unlikely to contribute significantly, because these compounds contain hydrogen and therefore react with hydroxyl radicals ( O H ) in the troposphere. A s a result, Brown estimates that they have lifetimes in the atmsophere of between two and six years.

Depletion of the earth's ozone layer by C F C s and other halogenated compounds results in an increase in ultraviolet radiation at the earth's surface. This is a direct threat to life. Increased radiation m a y cause skin damage and eye damage such as cataracts. It has been estimated that depletion of the ozone layer by 1% will cause an extra 70,000 cases of skin cancer world-wide.

Increased ultraviolet radiation also reduces the efficiency of the i m m u n e system of the human body. This will m a k e people more susceptible to various diseases, including parasite attacks. The increased radiation m a y also adversely affect plants and food crops as well as wildlife.

Contributions of C F C s to the greenhouse effect

Carbon dioxide (C0 2 ) , water vapour and other gases in the atmosphere allow solar ultraviolet radiation to penetrate and w a r m the earth's surface. However, these gases also absorb the infrared radiation emitted from the w a r m surface of the earth. C 0 2

absorbs radiation in the waveband 13-19 /an and water vapour in the band 4 to 8 /un.

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The heat trapped in this 'thermal blanket' around the earth keeps the earth warm and is known as the greenhouse effect. Even so, about 70% of the earth's radiation escapes into space through the waveband 'window' between 8 and 12 /mi . In this band, natural atmospheric gases absorb only weakly. Before the 20th century an equilibrium was established between the amount of energy lost back into space. The earth's temperature thus remained reasonably constant.

Over the last century, however, the combustion of fossil fuels and industrial activity have disturbed this equilibrium. C 0 2 levels in the atmosphere have increased from 280 parts per million (ppm) in 1860 to about 350 p p m in the 1980s. The increased amounts of CO2 have absorbed more and more infrared radiation and this has caused the earth's temperature to rise 0-5 + 0 1°C since the 19th century. With increasing temperatures, and therefore hotter summers and milder winters, glaciers will begin to melt and sea levels rise. Weather patterns m a y also change and crop yields m a y be affected.

Other anthropogenic gases, such as C F C s , dinitrogen oxide ( N 2 0 ) and methane (CH 4 ) have also increased the size of the thermal blanket around the earth. C F C s absorb infrared radiation in the waveband from 8 to 12/im, thus blocking the window through which infrared radiation used to escape. The natural balance is therefore upset even more. C F C s are very efficient greenhouse gases. The greenhouse warming effect of one molecule of C F C - 1 1 or -12 is equivalent to 10,000 molecules of C 0 2 . Calculations suggest C F C s cause about 15 to 20% of greenhouse warming.

Paradoxically, the greenhouse gases do have an advantageous impact on the depletion of ozone in the stratosphere. Since more and more heat is being trapped in the thermal blanket, the stratosphere is becoming cooler. Consequently the rate of ozone depletion is being slowed d o w n , because the rate decreases at lower temperatures.

The Montreal Protocol

Over the past two decades society throughout the world has become increasingly aware of several forms of global pollution that could potentially have a disastrous impact on life on earth: ozone depletion, greenhouse warming, acid rain and chemical smog.

In the middle 1970s, following the work of Rowland and Molina, national and international agencies began to recognize that C F C s might be amongst the major culprits in contributing to global pollution. In 1976 the Environment Protection Agency (EPA) in the U S A announced its intention to prohibit the non-essential use of C F C s as aerosol propellants. Even so, by 1984, production of C F C s world-wide had passed their pre-ban levels and exceeded one million tonnes annually.

O n 16 September 1987, 24 nations signed the Montreal Protocol produced under the auspices of the United Nations Environment Programme ( U N E P ) . The following are the three main requirements of the protocol:

The 1986 levels of consumption of CFCs-11 , -12, -113, -114 and -115 must not be exceeded after mid-1989.

The 1986 levels of production must be reduced by 20% from 1 July 1993 and an additional 30% (bringing the total to 50%) by 1 July 1998.

A freeze at 1986 levels of consumption of halons-1211, -1301 and -2402 by 1994

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M . Freemantle

M o r e nations agreed to the protocol at international ozone conferences held in London in March 1989 attended by representatives from 123 countries and in Helsinki in M a y 1989.

Alternatives to C F C s

With the production of C F C s scheduled to decline dramatically over the next decade or so, C F C producers are already investing heavily in the development and testing of environmentally friendly C F C substitutes and alternative technologies which will meet the needs of society. However, it is recognized that is many cases—refrigeration and air-conditioning equipment for example—large reductions in emissions, particularly in the short term, can be made simply by recovering and recycling C F C s during servicing.

Several companies are actively involved in the development of activated carbon systems for the recovery of C F C s used as blowing agents for plastic foams. The systems are expensive but m a y be able to recover 40% of the agents.

According to some industrialists it is likely that conservation of C F C s and utilization of alternative products or technologies will reduce the demand for C F C s by up to 60%. This will leave about 40% of the current market for substitution by C F C alternatives—a market of perhaps 500,000 tonnes per year.

Requirements for CFC replacements

Potential alternatives to C F C s need to fulfil several requirements:

They should have approximately the same basic physical properties as C F C s , for example similar boiling points.

They should, as far as possible, have the safety features of C F C s , for example non-flammability and low toxicity.

Eventual commercial-scale production should be technically and economically viable. Ideally, equipment manufacturers would like to have 'drop-in' alternatives, that is compounds that do not deplete ozone but behave as C F C s in use. This will avoid modifications to production lines.

They should have adequate chemical stability for envisaged applications but at the same time be less stable than C F C s in the atmosphere so that little if any reaches the stratosphere. They should thus have m u c h shorter lifetimes in the stratosphere and zero or very low potentials for ozone depletion and greenhouse warming.

It is perhaps an irony that stability, the very factor which made C F C s so important, n o w makes them a threat to the environment. Reduced atmospheric stability can be achieved by the presence of hydrogen in the molecule. This allows the molecules to be degraded by hydroxyl ( O H ) radicals.

Partially halogenated methane and ethane candidates—all containing hydrogen— are n o w under consideration as C F C alternatives (see table 2). H C F C s - 2 2 and -142b and H F C - 1 5 2 a are already commercially available; the others are undergoing tests.

Ideally, candidates for replacing C F C s should contain no chlorine. However, it m a y be impossible to find compounds covering the whole useful range without including at least some chlorine-containing compounds.

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Table 2.

Possible alternatives to CFCs.

Compound Formula Uses

Hydrochlorofluorocarbons (HCFCs): HCFC-22 HCFC-123 HCFC-124 HCFC-Hlb HCFC-142b

CHC1F2

CHC12CF3

CHClFCFj CH3CC12F CH3CC1F2

Hydrofluorocarbons (HFCs): HFC-125 CHF2CF3

HFC-134a HFC-152a

CH2FCF3

CH 3CHF 2

Refrigerant, blowing agent Refrigerant, blowing agent Refrigerant, blowing agent Propellant, blowing agent Propellant

Refrigerant Refrigerant Refrigerant, propellant

Figure 1.

Properties of C F C s depend on the relative numbers of hydrogen, chlorine and fluorine atoms in the compounds.

Toxic

Hydrogen

Flammable

Chlorine ¿a

Long atmospheric life

Fluorine

T h e properties of C F C s , H C F C s and H F C s can be compared by drawing a triangle

with hydrogen, chlorine and fluorine at the corners (see figure 1). T h e bottom line

represents C F C s . They have a long atmospheric life and low flammability. Toxicity

depends o n the amoun t of chlorine present. H F C s are represented by the right hand

side of the triangle. Their toxicity is low and their flammability depends on the a m o u n t

of hydrogen present. H C F C s can be located anywhere in the triangle depending o n the

proportions of hydrogen, chlorine and fluorine that are present. Their properties vary

accordingly.

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M . Freemantle

Environmental acceptability

In October 1988, the results of an 'Alternative Fluorocarbon Environmental Acceptability Study' (AFEAS) were presented at a U N E P meeting in the Hague. Fifteen international chemical companies contributed to A F E A S . The study was concerned with the ozone depletion potentials (ODPs) and global warming potentials ( G W P s ) of H F C s and H C F C s and in particular: H C F C s - 2 2 , -123, -124, -141b and HFCs-125, -134a, -152a.

The capacity of a compound to destroy ozone depends on the amount of chlorine it contains and also its lifetime in the atmosphere. O D P s measure the contribution of various C F C s and their possible replacements relative to C F C - 1 1 , which is given a value of 10. The G W P s were calculated using computer models and are also relative to C F C - 1 1 with a value of 10. S o m e of the values quoted by U N E P and A F E A S in their reports are shown in table 3.

The final A F E A S report, published in 1989, concluded that only H C F C s with long atmospheric lifetimes have any potential to affect stratospheric ozone and that the lifetimes are determined by their rate of decomposition in the troposphere. For example, A F E A S found that H F C - 1 3 4 a has an atmospheric lifetime of 15-5 years and its O D P is zero. HFC-134a has a small G W P of about 0-27. O n the same scale, the G W P of C F C - 1 2 is 31. Therefore, substitution of HFC-134a for C F C - 1 2 will reduce the global warming potential by at least 91%.

A F E A S called for more detailed work on: atmsopheric degradation pathways of H F C s and H C F C s ; environmental concentrations and fate of the stable products; and the biodégradation pathways for any compound unexpectedly present in concentrations that might be of biological significance.

A new consortium of C F C producers k n o w n as A F E A S II has since been established to conduct the second stage of the study. The programme will last three years and provide support and funding for research in industrial, academic and government laboratories on the atmospheric and terrestrial effects of a number of C F C replacements. The final results are expected in 1993.

Table 3. ODP GWP

Compound

CFC-11 CFC-12 CFC-113 CFC-114 CFC-115

HCFC-22 HCFC-123 HCFC-124 HCFC-141b HCFC-142b

HFC-125 HFC-134a HFC-152a

(relative to CFC-11 = 1-0)

10 10 0-80 10 0-60

005 002 002 010 006

0 0 0

(relative to CFC-11

10 2-8-3-4 1-4 3 - 7 ^ 1 7-5-7-6

0-34-0-37 0 0 1 7 - 0 0 2 0 0-092-0-10 0-087-0097 0-34-0-39

0-51-0-65 0-25-0-29 0-026-0033

Ozone Depletion Potentials (ODPs) and Global Warming Potentials (GWPs) of CFCs, HCFCs and HFCs.

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In January 1988, a Programme for Alternative Fluorocarbon Toxicity Testing (PAFT) was established to evaluate the safety of suitable C F C alternatives. The programme, which is parallel to A F E A S , was launched by 14 companies. P A F T I developed toxicity profiles of H C F C - 1 2 3 and H F C - 1 3 4 a . P A F T II was formed by ten C F C producers in September 1988 to study H C F C - 1 4 1 b and in June 1989 P A F T III was set up by seven producers to examine H C F C - 1 2 4 and H F C - 1 2 5 .

The results of studies on the acute, subacute and subchronic effects of H C F C - 1 2 3 , H F C - 1 3 4 a and H C F C - 1 4 1 b , together with in-vitro tests on their mutagenicity and teratogenicity, were encouraging and indicated that the toxicity profiles of these three compounds are very similar in m a n y respects to CFCs-11 and -12. Combined two-year studies on their chronic and carcinogenic effects are still continuing and the results will not be available until 1993 at the earliest. Studies on the P A F T III products H C F C - 1 2 4 and H F C - 1 2 5 are currently at a preliminary stage, with results expected in 1994-5.

Alternatives to CFC-11

C F C - 1 1 is used in refrigeration, industrial air conditioning and as a blowing agent. The compound acts as an excellent insulator and is non-flammable: it is, for example, used as a blowing agent for rigid foams which fill the outer walls of refrigerators.

It is technically possible to produce foam using water with carbon dioxide as the blowing agent, but the product is a poor insulator. Dichloromethane (CH 2 C1 2 ) can also be used but it is flammable.

The main contender to replace C F C - 1 1 in industrial systems is H C F C - 1 2 3 . This has only 2% of the G W P of C F C - 1 1 (see table 3). It m a y be synthesized from C C 1 3 C F 3 :

CCI3CF3 -» C H C 1 2 C F 3 + C H 2 C 1 C F 3

HCFC-123 HCFC-133a

However, there are a number of process difficulties with this method of synthesis. Trace impurities can cause degeneration of the catalyst and the products form azeotropes (constant boiling mixtures), which makes separation by fractional distillation difficult. Furthermore, H C F C - 1 3 3 a is toxic.

D u Pont, the American chemical company, is constructing a number of pilot and full-scale plants to produce H C F C - 1 2 3 . The product could replace C F C - 1 1 in industrial cooling plants and in the production of polyurethane and phenolic foams. Imperial Chemical Industries (ICI), a UK-based company, is currently carrying out research on H C F C s - 1 2 3 and -124b as alternative blowing agents to C F C - 1 1 , both being assessed in various formulations. H C F C - 1 4 1 b is another long-term possible alternative to C F C - 1 1 for the manufacture of foam insulation.


H F C - 1 3 4 a leads the field of candidates to replace C F C - 1 2 in refrigeration and air-conditioning applications, especially for automobile air conditioning, refrigerators and freezers, commercial building air conditioners and other industrial systems.

Since the properties of H F C - 1 3 4 a are similar to C F C - 1 2 it comes near to being a 'drop in' replacement. Like H C F C - 1 2 3 , the main problem lies in the manufacture. Most of the m a n y process routes for H F C - 1 3 4 a are three- or four-step processes and therefore costly.

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M . Freemantle

In 1990 ICI began production of HFC-134a in the U K and plans another plant for 1992 in the U S A . The company is already marketing a product with the trade name K L E A 134a. Results of P A F T studies show that K L E A 134a has a low level of toxicity—comparable to C F C - 1 2 .

ICI has patents on several processes for the manufacture of HFC-134a . O n e of these uses trichloroethene as feedstock and produces the toxic H C F C - 1 3 3 a in high yield as an intermediate:

HF HF C H C 1 = C C 1 2 • C H 2 C 1 C F 3 • C H 2 F C F 3 + HCl

HCFC-133a HFC-134a

The second step gives only a low yield of HFC-134a, and recycling is therefore necessary.

H C F C - 1 3 3 a can also be converted to HFC-134a using antimony (V) fluoride as a catalyst. Another possible route uses a chromium oxide catalyst and tetrachloroethene as feedstock. H C F C - 1 2 4 is an intermediate in this case. The use of CFC-114a (formula: C F C 1 2 C F 3 ) as a feedstock for H F C - 1 3 4 a is also currently being explored. CFC-114a is a by-product in the manufacture of CFC-114 .

D u Pont is operating a pilot plant to produce H F C - 1 2 4 in several new blends aimed at replacing C F C - 1 2 and other products in a broad range of refrigeration applications. O n e combination contains H F C - 2 2 , HFC-152a and H F C - 1 2 4 . The blend is a near azeotrope and has a 96% lower O D P and 91% lower G W P compared to C F C - 1 2 . A similar blend containing C F C - 1 1 4 is already available commercially.

C F C - 1 2 is used to produce polystyrene foam. The C F C is blown into molten resin to make packages for fast-food and also meat trays and egg cartons. D u Pont is marketing Formacel-S for this application. The product is a highly purified grade of H C F C - 2 2 , with a twentieth of the O D P of C F C - 1 2 .


The low toxicity, non-flammability and stability of CFC-113 has made it the ideal solvent for many applications. The electronics industry has been using C F C - 1 1 3 in increasing amounts over recent years for cleaning electronic assemblies and particularly for removing solder flux.

Water is probably the safest substitute but it poses regulatory and technical problems. Mixtures of alcohols, such as ethanol and propan-2-ol are promising substitutes and they are clean and readily available. A T & T and Petroferm Inc., in the U S A , have developed a substitute which is a biodegradable mixture of terpenes and surfactants known as Bioact E C - 7 .

There is possibly no single substitute for CFC-113 for cleaning electronic components. The Japanese electronics industry favours recycling CFC-113 to minimize its escape to the atmosphere. Some plants in Sweden avoid flux completely by the use of laser welding and electrically conductive glue instead of solder.

D u Pont has introduced two transition products ( F R E O N S M T and F R E O N M C A ) for solvent uses. These products have 25% and 37% lower O D P s respectively compared with CFC-113 . F R E O N S M T helps prevent white residue, eliminates ionic contamination and removes stubborn flux residues from printed wiring assemblies. It is

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a proprietary azeotrope of C F C - 1 1 3 , methanol and dichloroethene with a stabilizer package.

F R E O N S M T and F R E O N M C A are regarded as interim products in the transition to m o r e environmentally acceptable alternatives. D u Pont had announced two long-term candidates to replace C F C - 1 1 3 . O n e is a blend of H C F C - 1 4 1 b , H C F C -123 and methanol with a 90% lower O D P than C F C - 1 1 3 ; the second is a proprietary mixture of solvents and surface-active agents with zero O D P . •

Acknowledgements

Information used in preparing this article came from a wide variety of sources. In particular, the author gratefully acknowledges the help of: R . J. Baker (Director, British Refrigeration Association, Bucks, U K ) ; B . D . Joyner (Rhône-Poulenc, Avonmouth, U K ) ; V . T . Sheridan (Du Pont de Nemours International S.A., Geneva, Switzerland); C . E. Tane (ICI General Chemicals, Runcorn, U K ) ; the British Aerosol Manufacturers Association (London, U K ) ; and the United Nations Environment Programme.

News items, reports and articles in a wide variety of newspapers, magazines and journal also provided a wealth of information. The author is especially thankful to: Chemical and Engineering News, Chemistry in Britain, Nature and New Scientist.

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Reference materials: their role in measurement accuracy

W . P. Reed and S. D . Rasberry

Measurement compatibility and the establishment of production standards are very important aspects of manufacturing, material processing and environmental monitoring. Organizations such as the US National Institute of Standards and Technology play a key role in providing the wide variety of reference materials required.

For centuries, mankind has recognized the need for measurement compatibility of dimensional measurements. The cubit, as a standardized unit of length, was one of the earliest such recorded standards. Without this dimensional standard, it is entirely possible that the engineering marvels of 66 centuries ago, the Egyptian pyramids, could not have been built.

For various societies, dimensional units have changed over time, as is evidenced by such length units as the yard, the fathom, the furlong and the mile. Eventually, it was decided that the unit of length along with other quantities such as mass, time, temperature, and amount of a substance were defined in a coherent system of units, the Systeme international d'unités (SI), with definitions provided for each quantity. For example, in this system the unit of length, the metre, is defined as the distance light travels in a vacuum in a specified amount of time.

While the SI, 'metric system', provides chemical measurements, with the mole, kilogram, ampere, and Avogadro's constant, the 'rulers' needed to realize chemical measurements of constituent materials in a matrix are largely 20th Century developments. At the U S National Institute of Standards and Technology (NIST, formerly known as the National Bureau of Standards, N B S ) , the origin of reference materials dates to 1904, when the American Foundrymen's Society came to the newly formed N B S with a problem in the measurement of carbon and sulfur in cast iron. This

William P. Reed is Chief of the Standard Reference Materials Program of the United States National Institute of Standards and Technology (NIST), and as such is responsible for the 85-year-old program's development and administration. A graduate of Purdue University, Indiana, M r Reed has worked at the N I S T since 1964.

Stanley D . Rasberry graduated from Johns Hopkins University before joining the N I S T as physicist in 1963. H e is currently Director of Measurement Services there, with responsibility for policy development and general administration of four of the Institute's programs involving measurement and technology transfer— reference data, reference materials, calibration services and laboratory accreditation.

The authors m a y be contacted at the following address: U S Department of C o m m e r c e National Institute of Standards and Technology, Gaithersburg, M D 20888, U S A .


W. P. Reed and S. D. Rasberry

resulted in the issuance of a series of 'chemical rulers' then called 'Standard Samples' developed with the cooperation of chemists from the American Foundrymen's Society and intended to promote compatibility in the measurement of carbon and sulfur in cast irons. These measurements were metallurgically and economically important because the specifications by which cast iron was sold required certain concentrations for carbon and sulfur. The foundrymen k n e w that production of high-quality, durable cast iron depended on careful control of the metal composition to meet the specifications.

Following the development of this set of standard samples, other industries and trade associations requested the development of standard samples to control and harmonize measurements in their specific areas. Materials for these included steels, brasses, ores, limestone and sugars. Measurement compatibility of chemical measurements was indeed of commercial value and interest.

Standard Samples (now called Standard Reference Materials by N I S T ) play a leading role in the economical transfer of measurement quality. The certification laboratory can characterize an entire lot of material and then share that characterization with the whole measurement community by simply distributing individual samples from the lot to all of the laboratories involved. This economy of labor coupled with the ability to realize compatibility to national standards in one's o w n laboratory has m a d e Standard Reference Materials (SRMs) a popular and economical means of obtaining and verifying measurement compatibility.

Measurement compatibility is not an exact term but is used in the context of describing measurements that are in harmony with one another, or do not disagree with each other. That is, within the limits of measurement uncertainty, both numbers are equivalent.

Obviously, compatibility is very desirable in a measurement system and is a condition that most laboratories strive to obtain, for obvious reasons. It prevents disagreements among parties and helps promote credibility and product quality. W h e n compatibility is based on the national measurement system, it can provide valid evidence of compliance to environmental regulations, appropriate patient diagnosis, proper manufacturing and material processing. It is a key to productivity and helps preclude the expense of improper (bad) measurements.

Laboratories that certify Standard Reference Materials strive for accuracy to achieve measurement compatibility. Measurement accuracy, that is measurements without bias, automatically confers compatibility within the limits of the measurement process. For more details on the concepts of measurement accuracy and compatibility see the discussions provided by Cali and Reed1 and Taylor.2

For chemical S R M s , N I S T tries to achieve accuracy by two methods. O n e is the use of definitive measurement techniques. These are techniques that relate measurements to the SI system of units (i.e. mass, amperes, etc.), and have been extensively evaluated for potential measurement bias on a given matrix. In other words, the measurement process has been thoroughly studied for its ruggedness and potential bias and has been found suitable for the required measurements. The other method of certifying on the basis of accuracy is to use two or more independent and reliable measurement techniques. Here, the key is independence and reliability. This means that the measurement methods are completely different, employing different preparation and measurement approaches and that each method is k n o w n to produce reliable results. In this case, accuracy is demonstrated because the probability is low that measurements m a d e through completely different chemical processes would have identical biases.

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For S R M s , the measurement process is extensively evaluated for potential errors before the S R M is certified and two or m o r e independent sets of measurements are m a d e frequently in more than one laboratory. In any case, as part of the certification, the measurement technique itself must be well-defined.

Internationally, S R M s are called Certified Reference Materials ( C R M s ) under a definition3 provided by I S O - R E M C O . R E M C O is the Council Committee on Reference Materials of the International Organization for Standardization. ISO-R E M C O has also been responsible for assembling a directory of producers of reference materials4 and has collaborated in the development of a computerized search mechanism called C O M A R to identify Certified Reference Materials by element certified and matrix.

W h e r e enough C R M s of a given type are available, they can provide calibration points for industrial chemical analysis and other types of industrial measurement. For example, steel analysts employ N I S T C R M series for X-ray fluorescence analysis of low alloy steel, a relative analytical method in which good calibration standards are crucial.

Figure 1 shows two different ways of using Standard Reference Materials to assist measurement quality assurance. O n the left, the reference material has been used to determine the relationship between intensity measured in an instrument and concentration of an element in different materials.

S o m e instruments and methods have calibrations based on first principles. Here, calibrations m a y be based on pure element standards or binary mixtures of pure element standards. A very important role for C R M s emerges with the increased use of such methods—the role of method evaluation.

T o serve as a validator, the C R M is not used at all during the instrument calibration. Instead, it is measured along with the unknowns. It is placed on the graph as such and the results are compared with certified values to confirm or deny the validity of the measurement process.

M a n y countries n o w produce Reference Materials and Certified Reference Materials to meet their industrial and governmental needs. The following description of necessity covers those C R M s produced by N I S T . While not exhaustive, it does provide some indication of the kinds and varieties of materials currently being certified.

Figure 1.

The use of Standard Reference Materials.

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W. P. Reed and S. D . Rasberry

Metals and ores

As we indicated above, the metals and metal processing industries have been traditional users of S R M s for measurement and quality control. Consequently, a large number of reference materials developed over a long period of time are available in this area. For the ferrous industry, iron ores and reduced iron ore, as well as alloying elements such as ferrochrome and ferrosilicon, exist to cover the needs of the basic steel foundries. In addition, cast irons and a complete suite of low-alloy steels in solid and chip form are produced for the calibration of instrumentation used in the industry. Other compositions are also available for stainless steels, tool steels, speciality steels, and high-temperature alloys.

For the copper industry, copper ores and a variety of solid and chip copper materials are available, as are a number of brass compositions in solid form.

Aluminium producers provide a wide variety of alloy standards for optical emission spectrometry and X-ray fluorescence calibration (see figure 3). N I S T provides a variety of bauxite materials, a reduction grade alumina, and a series of benchmark aluminium alloy materials in both solid and chip forms that can be used to verify the accuracy of the measurement process.

A variety of other ores and 'minerals, as well as alloy compositions including lead and solder S R M s , are also certified by N I S T .

Spectrometric solutions

Atomic absorption plasma spectroscopy has become a major analytical tool in the chemical laboratory. Methods are based on the calibration of an instrument by a

Figure 2. Some of the many varieties of S R M s distributed by NIST.

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Reference materials

Figure 3.

Preparation of metal S R M s used for X-ray fluorescence and optical emission spectroscopy measurements.

material in solution, the spectroscopic characteristics of the sample material being shown as a graph or in figures. The extreme accuracy and simplicity of these methods have enabled them almost to replace classical methods. They have given rise to the need for solution standards. N I S T is issuing over 65 single-element and multi-element spectrometric solutions to meet the demand. The latter include a trace-element-in-water S R M containing 17 certified elements at the nanogram per gram level and a set of anion solution S R M s for electroconductance and acid rain research.

Environment

Standard Reference Materials provide a c o m m o n base for all levels of government that monitor pollution, and for industries that must control the emission levels of pollutants. A wide variety of materials are available for this purpose, including solids, liquids and gases certified for trace metals and organic pollutants such as P A H s and P C B s . A m o n g these are river and estuarine sediments, urban dust and chlorinated pesticides in iso-octane (figure 4).

Environmental gases are available in a range of concentrations for evaluating pollutants from both mobile and stationary power sources, as is a series of carbon dioxide in air S R M s used to monitor C 0 2 build-up in the atmosphere.

T o tackle the problem of measuring potential pollution a number of hydrocarbon fuels have been prepared. These include coals and fuel oils certified for trace elements and sulfur content. S o m e of the coal materials also provide heat content and 'proximate analysis' values (moisture, volatile matter, fixed carbon and ash). A fly ash typical of coal-burning power plants is also available to round out the series.

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Figure 4. Several of the S R M s used by scientists to accurately measure environmental contamination.

Clinical and nutritional areas

The first clinical S R M s were issued by N I S T about 20 years ago and marked a major expansion of the S R M program. These materials provide an accurate base for the clinical chemist. The currently available S R M s are used to develop 'reference methods' employed by manufacturers and clinical research facilities.

Materials of interest that are n o w available include a freeze-dried h u m a n serum, a series of cholesterol in serum materials (freeze-dried and frozen), a bovine serum albumin, anti-epilepsy drugs in serum and anticonvulsant drugs in serum. Also available are thermometric standards and a number of clinical materials certified for purity.

In the area of nutrition S R M s , several food and food-related materials certified for trace elements have been issued. These include dry powdered milk, wheat flour, and oyster tissue. Recently a coconut oil was certified for cholesterol and several vitamins and provides in effect a standard 'mixed diet' food material (figure 5).

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Reference materials

Electronics

Electrical resistivity is an important quantity used in the electronics industry as a predictor of material performance. S R M s are available for metal resistivity as well as for silicon resistivity.

Quality assurance for the photomasks that are used in producing integrated circuit microchips requires accurate measurement of the size of each feature on the photomask. N I S T has been producing linewidth measurement standards to calibrate the optical microscopes used to measure the widths of opaque lines and clear spaces on integrated circuit photomasks.

Engineering materials

M a n y kinds of materials used in testing the engineering properties of materials are certified at N I S T . Tire makers and others in the rubber industry have available a number of rubber and rubber-compounding materials. S R M s for surface area of powders, sieve size, particle size, and cement fineness and composition are also issued.

Several S R M s are of use in construction and in fire research. For example, some are used to calibrate smoke-density chambers. Others are useful in calibrating devices which measure the heat insulation qualities of building materials.

Figure 5. This coconut oil S R M is certified by NIST for cholesterol and vitamins A , D and E .

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The future

The decade of the 1980s was a time of rapid growth in the development of reference materials in m a n y parts of the world. It was a time of remarkable achievements by hundred of researchers, pushing the limits of quantitation below the part per billion level, spatial resolution below the micrometer level, and the homogeneity of materials to previously unrealized uniformity.

In the People's Republic of China alone, more than 500 new reference materials have been developed in the course of the last ten years. At the same time, there has been increased interest in Europe and the Americas. The 1980s even saw reference material production aboard the space shuttle Challenger under microgravity conditions. Despite all these remarkable events, the next decade promises to include even wider-scale production, certification and use of reference materials. Four trends should be especially notable in this period:

Globalization

The stage is set for more coordination (if not outright cooperation) in the development of reference materials. There are at least two underlying reasons for this. Frequently, reference materials are urgently needed, but by only a few specialists around the whole world. The demand m a y be so small (one or two units per year) in even the largest countries that no one program can afford the resources to respond. In some cases, producers will link their resources, across national boundaries, to share the cost of answering such needs. In other cases coordination m a y be provided by an organization such as I S O - R E M C O to steer entire markets to a single producer.

A second reason for more globalization in reference materials is purely h u m a n . People, especially scientists, are curious about new developments regardless of geographic borders. N I S T , for example, has had scientific exchanges on reference material development with at least ten countries in the past ten years, and it is difficult to see this not increasing.

Instrumentation

Here the trends seem very clear. Measurement methods of every kind will become more instrument-oriented and more automated. Usually, instrumental methods are comparative or relative measurements and require the use of calibration and validation materials. Thus, it is likely there will be a general increase in the kinds and quantities of reference materials needed. Clearly, too, real-time process control based on in-situ chemical measurement will expand rapidly in the 1990s.

Laboratory accreditation

Increasingly during the 1990s, systems of accrediting laboratory capability and performance will be developed. Specialized commercial laboratories have increased services to support customers seeking to improve product quality, find equity in commerce, meet regulations, or attain other objectives. N o w customers of such laboratories are searching for ways of ensuring that the measurement information which they receive is of high quality.

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Reference materials

O n e possibility for meeting customer requirements is systematic assessment, monitoring and accreditation of laboratory performance by a government agency or other 'disinterested, third-party' accrediting organization.

Experience has shown that accrediting organizations find certified reference materials to be very useful in testing laboratory performance. The materials can be used as part of a performance test, or they can be used on a routine basis by the laboratory to keep their measurement calibrations 'tuned up' and validated. A s these formal programs expand, there will be increased demand for greater variety and availability of reference materials.

Frontiers

It is easy to guess that m a n y techniques n o w being tested in a research setting will become commercialized in the instruments of tomorrow. The list can start with new sampling devices and extend through automated sample processing, through new resolution techniques, to new detection, discrimination and data processing systems. A s one tool has reached its physical limits of refinement, for example, magnification in optical microscopy hitting light diffraction limitations, so new tools have emerged from the laboratory. The 1990s will undoubtedly see new S R M s required for resolution, magnification and microanalysis in optical, electron, and perhaps even electron tunnelling microscopes.

Frontiers in microanalysis will be pushed back, and so will those in trace element analysis. Determination of essential as well as toxic elements and their organic forms will be carried out routinely at part-per-billion and part-per-trillion levels by the end of this decade. The bigger question here (and for a wide range of organic compounds, too) will be what are the actual health effects? W e are likely to see development of highly portable analysers and monitors for biological fluids to help researchers assess exposures and effects. W h a t a challenge this m a y present to future producers of reference materials!

'Prevention' is a popular medical buzzword today. In the 1990s it will be increasingly applied to aging engineered systems such as aircraft, oil tankers, bridges, highways and water supplies. Failure prevention measurements such as X-raying metal welds and the discovery of cracks in aircraft parts will not only help us to make safe use of expensive systems, but also to accurately judge the end of their service life. Physical and engineering tests of materials and systems are currently not as well supported by reference material availability as are chemical tests. Thus, the need for more such S R M s in the coming decade is clear. •

References

1. C A L I , J. P., and R E E D , W . P. (1974). The Role of the National Bureau of Standards Standard Reference Materials in Accurate Trace Analysis, N B S Spec. Publ. 422, Accuracy in Trace Analysis, Sampling, Sample Handling, and Analysis.

2. T A Y L O R , J. K . (1985). Handbook for SRM Users, N B S Spec. Publ. 260-100 (1985). 3. ISO Guide 30—Terms and Definitions Used in Connection with Reference Materials; 1981.

Available from A N S I , 1430 Broadway, N e w York, N Y 10018, U S A . 4. ISO Directory of Certified Reference Materials: 1982. Available from A N S I , 1430 Broadway,

N e w York, N Y 10018, U S A .

79

Chemicals through biotechnology: facts, hopes, dreams and doubts

Chandana Chakrabarti and Pushpa M . Bhargava

Thanks to the remarkable progress made in genetic engineering and cell culture techniques in recent years, we are now in a position to persuade living systems to do jobs that they have never done before. Microbes and plant and animals cells are being exploited to produce an already wide range of useful chemicals, and the prospects for major new developments in this area of biotechnology are good.

Historical imperatives operate as m u c h in science as they do in other areas of h u m a n endeavour. The development of technology has been such an imperative through history. Whenever m a n has acquired a reasonable understanding of the nature, structure and function of a naturally-occurring system, he has inevitably been moved to use this knowledge for practical purposes. This was the basis of the development of chemistry-based technologies that led to the Industrial Revolution of the 17th and the 18th centuries, the physics-based technologies in the earlier part of this century, and the astrophysics-based technologies—that is, space technologies—in the latter part of this century.

O f the four fundamental basic sciences, biology was the last to develop. However, since the early 1950s and the determination of the structure of D N A , there has been an unprecedented explosion of knowledge in the area of biology. It has been as if, all of a sudden, m a n y pieces of a large jig-saw puzzle have fallen into place and a general picture has begun to emerge. It was, therefore, predicted in the late 1960s by those w h o had foresight and could take a global view of the changes that were taking place in modern biology that, sooner or later, this new knowledge was bound to find practical application. It happened sooner than expected: the emergence of biotechnology was nothing less than spectacular.

Chandana Chakrabarti is Communication Officer at the Centre for Cellular & Molecular Biology ( C C M B ) , the premier Indian national institution for research in molecular biology, located at Hyderabad.

Pushpa Bhargava, the founder-Director of the C C M B , is a widely known, highly cited and much honoured molecular biologist who has played a key role in the development of modern biology and biotechnology in India.

D r Bhargava and Mrs Chakrabarti can be contacted at the following address: Centre for Cellular and Molecular Biology ( C C M B ) , Hyderabad 500 007, India.


C. Chakrabarti and P. M. Bhargava

O u r limited objective in this article is to provide a flavour of what has already been done with regard to the production of chemicals through biotechnology; what is on the anvil—that is, where m u c h basic research has already been done and, therefore, where it is likely that applications would emerge in this century or, say, during the first quarter of the next century; and what are the unanswered basic questions that would determine the production of future generations of chemicals through biotechnology. Finally, w e shall briefly look at the problems that large-scale biotechnological production of chemicals is likely to throw up.

What has been and is being done

The contribution of genetic engineering technology

Genetic engineering involves altering or modulating the genetic—that is, the inherited—potential of a living cell or organism by introducing into it foreign genetic information, for example for making materials which the cell or the organism is otherwise incapable of producing. This technology has various advantages.

If the host cell receiving the foreign genetic information is a microorganism such as E. coli or a yeast which can grow very rapidly, one can produce large quantities of materials which are normally found only in extremely small amounts, say, in animals, including h u m a n beings, or plants. A n example would be h u m a n insulin—the drug traditionally used for diabetes—which has been virtually unavailable; the insulin commercially available so far, therefore, has been insulin from pig or cattle. Although the animal insulins are effective in controlling the major symptoms of diabetes, they have some harmful effects, for example on kidney and retina; some individuals are also allergic to animal insulin.

Reactions in biological systems occur through the intervention of enzymes which serve as biological catalysts. A substantial number of enzymes being currently exploited are from plant and animal (including h u m a n ) sources. For example, the enzyme cholesterol oxidase is employed to monitor the level of cholesterol in blood serum, and the enzyme uricase serves to monitor the level of uric acid. A n d there are enzymes used in brewing, baking and meat tenderizing, and in the manufacture of detergents and leather; the enzyme rennin is used in cheese making. The problem of using enzymes on a large scale has been that they are often unstable. It is possible today to genetically alter the gene for the enzyme in such a w a y that it becomes inherently stable; success has already been achieved in stabilizing the protein fibroblast growth factor (FGF)—and all commercially useful enzymes of today are proteins—by replacing one of its lysines by serine. Similarly, through genetic engineering, it is possible to shift the p H optimum of an enzyme or to modify its substrate specificity or kinetic parameters. It has already been possible to do so in the case of the enzyme subtilisin (which catalyses the breakdown of proteins), by changing the charge on the surface of the protein through the n e w technique of site-directed mutagenesis.

Genetic engineering technology allows minor modifications to be m a d e in enzymes so that their therapeutic effects are enhanced. For example, a monomeric insulin obtained by genetic engineering is more easily absorbed.

O n occasions, there is need only for transient synthesis of a particular protein in the given tissue of an animal. It has been possible to introduce and express a h u m a n insulin

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Chemicals through biotechnology

gene in adult rat liver, using artificial lipid vesicles—the liposomes—to package the insulin gene; the liposomes can be made in such a way that they go only to liver and, by fusion with the liver cells, transfer the gene to these cells where the gene is active for a while and then decays.

So far, recombinant D N A technology has allowed the production of m o r e than eighty proteins by microbial cells or animals which normally do not produce such proteins. S o m e of these cloned proteins are already commercially available; others are in various stages of development, production, trial or investigation. These proteins have a variety of uses such as therapeutic agents for inflammation, burns and w o u n d -healing, various types of malignancies and infections, arteriosclerosis, arthritis, ulcers, blood clotting or digestive disorders, hypertension, pain, haemophilia A , osteoporesis, aplastic anaemia, fertility control, diabetes, keratitis, hepatitis B , autoimmune diseases, diseases of the eye, myocardial infarction, thromboses, certain metabolic disorders, pancreatitis, gastric bleeding, and control of blood pressure. They are also used as plasma expanders, surgical adhesives, laboratory reagents, adjuncts in tissue transplantation, analytical reagents, agents for increasing milk yield or body weight of fish, and as anti-coagulants.

There have also been indirect uses of genetic engineering technology. In Australia, feeding sheep with a diet containing genetically engineered alfalfa that has an increased content of cysteine, has led to a 5% increase in production of wool, worth Australian $300 million in a single year. A n d all this has happened in a decade-and-a-half since the pioneering work of Paul Berg, for which he received the Nobel Prize for Chemistry in 1980.

The new vaccines

Advances in biotechnology have opened up a new approach to the development of vaccines, using antigens that have been produced through genetic engineering. This technique allows a modification of the naturally occurring antigens to remove some of the disadvantages inherent in the natural antigen, such as instability and possibility of contamination, for example, in the case of viral antigens, with incompletely inactivated virus. Moreover, in the case of a natural antigen, sufficient amount of the i m m u n o g e n is often not available—as, for example is the case with the hepatitis or the foot-and-mouth virus, or with Mycobacterium leprae, the leprosy bacillus.

For the foot-and-mouth virus disease of cattle, a vaccine m a d e through the use of recombinant D N A technology has already been available in the West for s o m e time. Another recent pioneering success has been with the hepatitis B vaccine. In France alone, the vaccine has been tested on several thousand volunteers, and has n o w been approved for h u m a n use; it contains the pure viral surface antigen which, on secretion from a genetically manipulated yeast, spontaneously forms aggregates that substantially resemble the natural virus shell, and are highly immunogenic.

Scientists at Transgene, in collaboration with Institut Pasteur in Paris, have come close to developing a cheap vaccine for schistosomiasis, which affects about 250 million people in the tropical regions of the world every year. There has been progress in genetically engineering a vaccine for dengue fever, a severe viral disease that is beginning to spread n o w also to North America; it is mosquito-borne and has been so far endemic to m u c h of Asia, South Africa, and South and Central America. The cloning of gene for the major surface protein oí Plasmodium falciparum, the species that

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causes most of the severe malaria infections in h u m a n s , has opened the way to an effective anti-malarial vaccine that would, hopefully, prevent malaria in the vaccinated individual and block its spread; the vaccine has undergone trials, for example in Colombia.

About 12 million people suffer from leprosy. In India alone, there are about 300,000 new cases each year, and millions of Indians will require vaccination if the country is to meet its target of eliminating the disease by the year 2000. A cloned vaccine, however, could provide the quantities needed to eliminate leprosy altogether. Scientists are n o w well on the way to genetically engineering a vaccine for leprosy. They have identified antigens on the surface of Mycobacterium leprae and have already cloned some of the antigens in E. coli; they are n o w in the process of determining which of these antigens would be suitable for a vaccine. S o m e work m a y also provide a better diagnostic test for leprosy than those available n o w . Three leprosy vaccines, two developed indigenously and one under the auspices of W H O , are already under trial in India.

There has been also significant progress on four of the five vaccines against agents that cause diarrhoea: rotaviruses which are responsible for up to 40% of the life-threatening diarrhoeas in children under two years of age, and to 500,000 deaths per year; Salmonella typhimurium, the agent of typhoid fever; cholera; and Shigella dysenteriae. The rotavirus vaccine developed by D r Albert Kapikin of the U S National Institutes of Health, is under trial in several countries such as Finland, Venezuela and Peru. For some inexplicable reason, success with the genetically engineered cholera vaccines has been limited. Other diseases for which vaccines based on antigens produced through genetic engineering are being attempted and are in various stages of development and/or trial, are A I D S , influenza, meningitis, herpes, rabies, pertussis (whooping cough), measles, poliomyelitis, tuberculosis, rheumatic fever, pneumococcal infections, and the rinderpest disease of cattle. W H O has a major action programme on vaccines.

Vaccines designed to control fertility, based on genetically engineered products such as the beta chain of the h u m a n choriogonic gonadotrophin, are also under investigation or clinical trial. With the inherent advantages of a genetically engineered vaccine, success with any one of the above-mentioned vaccines will be a landmark in the history of control of disease on our planet.

Immunotechnology-based chemicals and the new R N A technology

W h e n a foreign substance enters the body of a vertebrate animal, a large number of a class of protein molecules called antibodies are secreted by plasma cells (one kind of white cell) of the blood; this response of the body is called the i m m u n e response. Antibodies have the ability of combining with the antigen, i.e. the foreign substance, and setting in train processes that can neutralize and eliminate the foreign substance. T h e antibody response to a typical antigen is highly heterogeneous, one plasma cell clone in blood producing only one type of antibody. Thus , as each antigen leads to the proliferation of a variety of plasma cells, it also leads to the production of a variety of antibodies, each different chemically. The pioneering work of Georges Köhler and César Milstein, for which they received the Nobel Prize for Physiology or Medicine in 1984, has n o w m a d e it possible to select and immortalize plasma cells of one particular type producing just one chemical species of antibody, by fusing them with a tumour

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cell. Each plasma cell clone thus formed becomes a long-term source of substantial quantities of a single, highly specific antibody. The individual hybrid cells can be maintained indefinitely and, at any time, frozen samples of the cells can be grown in culture or injected into animals for the large-scale production of what is k n o w n as a 'monoclonal antibody' ( M A B ) . Highly specific M A B s produced by this general method have proved to be a remarkably versatile tool in m a n y areas of biological research and clinical medicine.

Monoclonal antibodies which can be produced in large quantities show promise of wide use as n e w reagents that would allow the rapid diagnosis of diseases. There will be home-diagnostic kits available for detection of pregnancy and of a variety of diseases, including cancer, at an early stage. Today, most types of cancer can be controlled, if detected early enough. In fact, it is probable that, in the next decade, learning to use such kits, and those that are likely to become available for the quantitative estimation of parameters such as sugar in urine, will be a part of the regular mandatory syllabus in schools. Home-testing for pregnancy is already a widely used—and perhaps the best-known—domestic application of biotechnology.

Monoclonal antibodies attached to radioisotopes injected into the body have improved radioisotope imaging techniques for cancer patients. Molecular conjugates of M A B s with cytotoxins such as ricin, radionuclides or gelonin, have the potential for selectively targeting themselves on the tumour cells and killing them; some of these conjugates, a type of immunotoxin, are undergoing initial clinical trials. Monoclonal antibodies m a y also be used to block specific cell receptors so as to inhibit the biological function of the particular cell type—for example, of T-cells that are responsible for the rejection of a tissue or organ transplant.

Recently, chimeric MABs—hal f mouse and half human—have been produced on a large scale as substitutes for completely h u m a n M A B s that would be ideal to prevent i m m u n e response in patients but that are difficult to produce directly. It is estimated that M A B technology will lead to revenues exceeding U S $2 billion per year by the end of this year.

As of today more than 200 immunodiagnostic kits developed for detecting diseases are available. Recently, Metlabs Ltd., of Dublin, have announced the release of a new E L I S A (enzyme-linked immunosorbant assay) test kit to measure a specific blood protein that m a y be a marker for high-risk smokers.

Perhaps the most exciting advance in immunotechnology in the last year or two has been the demonstration that both Escherichia coli and plants can be persuaded to produce antibodies, and that antibodies can also serve as enzymes, for example as a proteolytic enzyme that will break or cleave proteins at a specific site. These discoveries give totally new dimensions to the potential production and use of antibodies.

A Nobel Prize was recently awarded for the remarkable discovery that R N A (ribonucleic acid) can also serve as an enzyme. Viable methods have n o w been developed for the chemical synthesis of this nucleic acid and there is no doubt that there will be a spurt of research activity in the possible use—eventually commercial use—of R N A for carrying out specific enzymatic reactions of commercial importance.

Probes for diseases and D N A fingerprinting

The medical geneticist's bible, Victor McKusick's Mendelian Inheritance in M a n , lists some 3500 h u m a n diseases due to defective genes. Diseases such as haemophilia, cystic

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fibrosis and D o w n ' s syndrome are a m o n g the c o m m o n genetic disorders. Added to this, genetic factors m a y contribute to some congenital malformations and cases of mental retardation, and to some c o m m o n adult disorders such as diabetes, schizophrenia and coronary artery disease. In particular, the various inherited anaemias, such as sickle-cell anaemia and thalassaemia, affect hundreds of thousands of children in Africa, the Mediterranean region, the Middle East, the Indian sub-continent, and throughout southeast Asia. M a n y inherited diseases are not treatable; even if therapy is available, it is often expensive and painful and must be continued throughout life. Hence, m u c h effort has been put into their prevention. O n e of the exciting applications of recombinant D N A technology is likely to be the cure of h u m a n genetic diseases through correction of the 'mistake' in D N A structure that is responsible for the disease, or by transferring a normal functional gene into the defective cells. Accurate and timely genetic counselling will be one of the prerequisites for the use of recombinant D N A technology for combatting inherited disorders. Fortunately, it is n o w possible to diagnose m a n y such disorders very early in pregnancy, making it possible to terminate pregnancy if the foetus is found to have inherited a defective gene.

Researchers at the University of Arizona have developed probes to detect viruses in water. The test is so sensitive that it can trace ten virus particles in 1000 litres of water, and the probes developed can detect viruses that cause polio, meningitis and childhood diarrhoea.

O n e of the remarkable and useful applications of biotechnology has been in regard to the development of the technique of D N A fingerprinting. This technique of identification using the analysis of the genetic material of the individual is, today, the most sensitive and reliable means available for individual identification. It is based on cutting the D N A of an individual—and D N A obtained from just one hair root would be enough—with chemical scissors called 'restriction endonucleases', and separating the D N A fragments thus obtained on a gel by electrophoresis. The separated fragments are then probed with a suitable D N A probe genetically cloned in bacteria and thus prepared in large amounts by genetic engineering techniques; the probe is m a d e radioactive or otherwise suitably labelled before use. The pattern obtained on the gel on which the D N A fragments have been separated, following the above probing and autoradiography to detect the fragments of D N A to which the probe has stuck, is unique for the individual.

The success of this technique depends on the sensitivity of the probe. Today, two D N A fingerprinting techniques are commercially used—one developed by Alec Jeffrey in the U K , and the other developed by Lalji Singh and his colleagues in our laboratory at Hyderabad. There is little doubt that D N A fingerprinting will be widely used in the future, not only for identification of h u m a n beings and the establishment of relationships between them, but also for the identification of the germplasm of plants and appropriate documentation in this regard, and of animals in order to unambiguously define their lineage. Production of probes for this purpose, as for diagnosis of inherited diseases, would then become a major industry.

Chemicals from cell culture

O n e of the most important advances in terms of techniques that has revolutionalized modern biology is tissue culture—that is, the ability to m a k e both plant and animal cells grow and divide, somewhat like microbial cells, in laboratory glassware. In the

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case of a plant, for example the carrot, the entire organism can be regenerated from a single cell derived from any part of the plant. M a n y cells, both plant and animal, w h e n grown in tissue culture, produce and secrete into the medium chemicals of commercial importance. T h e cells can even be kept in continuous culture and the medium harvested from time to time and replenished. The m e d i u m can then be processed to obtain the desired chemical. In this manner, a fair number of chemicals of commercial significance have been produced in viable yield by plant cells grown in culture. S o m e of these substances are: ajmalicine, an anti-hypertensive agent; codeine, an analgesic; digoxin, a cardiatonic; diosgenin, an oral contraceptive; ubiquinone-10, used in treatment of heart diseases; berberine, an antiseptic; shikonin, used for treatment of burns and skin diseases, as a red pigment for dyeing silk, and in the manufacture of bio-lipsticks; anthocyanins, used as pigments; stevioside, a sweetner; and pyrethrum, an insecticide. Mexican researchers have developed a process for obtaining capsicum flavouring-agents using plant cell cultures. A n d it has been possible to grow the juice sacs of citrus fruits in laboratory dishes!

The use of animal cell cultures for producing industrially important chemicals has been more limited. Perhaps the most significant product obtained so far from cultured animal cells has been interferon of various types (alpha-1, beta-1 and beta-2) from cultured fibroblasts. It has been possible to immortalize rennin-producing cells so that there is a possibility of making this commercially important enzyme used in the manufacture of cheese from animal cells grown in tissue culture.

Exploiting the biochemical activities of microbes

Perhaps, the oldest biotechnological process for the production of a chemical k n o w n to m a n is the fermentative production of ethanol, or c o m m o n alcohol. Ethanol is one of the most important industrial organic chemicals and a potential major source of energy. Ethanol is employed as a solvent, an extractant and an antifreeze. Moreover, it is the starting substance for the synthesis of a large number of other organic compounds that serve as solvents, extradants, dyes, pharmaceuticals, lubricants, adhesives, ditergents, pesticides, plasticizers, surface coatings, cosmetics, explosives, and resins for the manufacture of synthetic fibres. Ethanol can be m a d e either synthetically from petrochemical feedstocks, or biologically from sugar sources such as molasses by the yeast Saccharomyces cerevisiae or other microorganisms.

Amongst the other chemical uses of fermentation is the production of acetic acid, acetone, butanol and citric acid. Citric acid, for example, is m a d e by moulds under conditions where a nutrient imbalance is created by limiting the supply of certain minerals such as iron and manganese; fermentative production from an appropriate sugar source such as molasses is the only source of this important chemical, without which our soft-drink industry m a y not be viable.

Other industrially important products that have been produced by microorganisms before the advent of the age of modern biotechnology include other organic acids, antibiotics such as penicillin, vitamins, and other secondary metabolites such as toxins and alkaloids. Then there have been algae-derived products such as the three phycocolloids—alginates, carrageenans and agar—without which m o d e r n life might well be impossible. These substances are used as gelling agents, stabilizers and emulsifiers in milk products, baked goods, toothpaste, shampoos, dyes and paints, and in paper and textiles and processed foods.

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C. Chakrabarti and P. M . Bhargava

S o m e of the m a j o r efforts in m o d e r n b iotechnology h a v e b e e n in the following directions:

(a) T o use non-conventional sources such as cellulose a n d related polymers of w o o d that are abundant , renewable a n d inexpensive, as the starting material for the production of ethanol. Plastics, such as polypropylene, a n d polyethylene are m a d e by the polymerization of alkene oxides which are n o w synthesized from petrochemical feedstocks. These plastics will b e c o m e m u c h cheaper if ethanol b e c o m e s m o r e a b u n d a n t and cheap. Therefore, a m a j o r effort in this area has been in the direction of development of enzymes that will break d o w n cellulose to its simpler sugar component s which can then be used by a microorganism to m a k e ethanol.

(b) T o find newer a n d cheaper biological sources of materials such as agar for which d e m a n d has increased tremendously in the recent past. T h e unprecedented use of agar in gel electrophoresis, a corner-stone technique in biotechnology, has led to a m o r e than ten-fold increase in the price of highly purified agar, that is, agarose, during the last few years.

(c) T o find n e w organisms that will give better yields of k n o w n products. F o r e x a m p l e , University of Florida researchers have recently claimed to h a v e created a bacterium that converts vegetable a n d w o o d sugars into ethanol with an efficiency of 9 0 - 9 5 % , o n the basis of sugar in the biomass .

(d) T o find n e w processes for the production of k n o w n chemicals. F o r e x a m p l e , M e x i c a n researchers h a v e recently developed a process for producing penicillin by solid fermentation which, being a non-sterile process, is economically advantageous; this process is claimed to p r o d u c e 17 times m o r e penicillin than conventional m e t h o d s , a n d in a shorter time.

(e) T o find n e w e r uses for products already k n o w n to be m a d e b y microorganisms. These products include anti-cancer drugs, anti-fungals, cardiovascular drugs a n d antivirals, for w h i c h there is a potential m a r k e t of over U S $600 million per year.

(f) T o c o m b i n e genetic a n d environmental manipulation to achieve hitherto un imag ined levels of conversion of the starting material to the desired pr imary metabolite under less stringent conditions.

A m a j o r effort in n e w biotechnology has been to either exploit unusual microorganisms or to find or engineer n e w ones that w o u l d p r o d u c e the desired material in yields a n d under conditions that are commercially exploitable. Let us look at s o m e examples .

Microorgan i sms are n o w being increasingly used to produce e n z y m e s . Candida rugosa lipase c a n separate the products of hydrolysis of tallow—the fatty acids, a n d glycerine—into t w o layers within 2 0 minutes at 4 0 ° C , which is orders of m a g n i t u d e faster than existing processes. There are microorganisms k n o w n today which c a n survive under conditions of considerable stress such as high temperature. Exploitation of the biochemical abilities of such organisms c a n be of considerable e c o n o m i c significance. F o r example , a n e n z y m e has b e e n discovered in the bacterium Acidothermus cellulolyticus, a resident of the Yellowstone hot springs in the U S A , w h i c h can be used for clarification of fruit juices even at 7 5 ° C . A pectinase derived from Aspergillus niger, can be used to obtain increased juice yields, reductions in viscosity, increased colour extraction, a n d enhanced clarification. Bacillus stearothermophilus has a lipase that can help b r e a k d o w n fats into smaller molecules for use in


confectionery. Its working temperature, 65°C, kills other microorganisms, thus sterilizing the product; fats liquify at this temperature, making solvents unnecessary. This saves m o n e y and avoids hazards from solvent residues. Other enzymes that are being or are likely to be produced on a commercial scale by microorganisms are amylase, streptokinase, lipase from Aspergillus which can be used as a detergent, protease from Thermonospora fusca which has a detergent action at 80°C, and aminopeptidase used in food processing. Reacting inulin with a refined inulin-decomposing enzyme from a certain type of soil bacteria, allows difructose, a well-k n o w n sweetner, to be produced in high yields.

Simple organic chemicals that are being or are likely to be produced in the immediate future microbiologically, starting from alkanes, include amino acids such as glutamic acid, ornithine, phenylalanine, tyrosine, phenylglycine, and hydroxyphenylglycine; organic acids such as alpha-ketoglutaric, fumaric, and long-chain ( C 1 2 - C 1 8 ) dicarboxylic acids; vitamins such as riboflavin, pyridoxin hydrochloride and pyridoxal phosphate, and Vitamin B 1 2 ; and several other substances such as ergosterol, beta-carotene, xanthophylls, and carbohydrates of various types. (The colloidal substance, xanthan, m a d e by the bacterium Xanthomonas campestris, is widely used as a food stabilizer and thickener; the petroleum industry is n o w beginning to include it as an ingredient of drilling muds.)

W e n o w have the capability to culture the algae Dunaliella salina in a manner that over 50% of its dry weight is glycerol. Similarly, the Japanese National Food Research Institute has succeeded in producing erythritol by fermenting grape sugar using Aureobasidium yeast, the yields being 47% of the sugar content. Erythritol might be useful as a non-caloric sweetener, since it is not metabolised by either the h u m a n body or the enteric bacteria; its sweetness is 80% that of cane sugar. In the near future it should be possible to produce chemicals such as ethylene, propylene, butylène and butadiene using fermentation processes and appropriate microorganisms. Biomass resources can also provide feedstocks to the chemical industry at reasonable prices, while conserving forest resources. Animal and plant wastes and sewage can be digested by bacteria and fungi to produce gas which contains about 60% methane. Methane biogas can be used for cooking and heating, or can be further converted to methanol (a liquid fuel). City garbage, too, can be used for the production of gas and electricity.

There are at least two reports of the production by microorganisms of biodegradable plastic material. A Japanese group has used hydrogen-fixing bacteria to convert 4-hydroxybutyric acid to polyesters which accumulate in large amounts inside the bacteria under the appropriate culture conditions. Biopol is another biodegradable polymer containing polyhydroxybutyrate produced by certain bacteria through a fermentation reaction; this polymer can be moulded and extruded in the same way as the conventional thermoplastics. Biopol is also compatible with animal tissues and thus has m a n y possible veterinary and h u m a n applications.

Plant- and animal-derived complex products

N e w techniques of purification have m a d e it possible to purify proteins such as factor VIII from blood, to a stage at which they can be commercially used. However, there is no question that they would be prepared m o r e economically through genetic engineering techniques. O n the other hand, n e w complex materials that are likely to find wide application continue to be discovered in the biological world that surrounds

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us. For example, a resin from the guayule plant which can serve as a source of natural rubber, appears to be a good potential agent for providing protection against various marine and terrestrial w o o d destroyers. A n d the strong natural glue, spore-tip mucilage ( S T M ) that can bind loose fungal spores of Magnaporthe grísea to various parts of rice plants that then succumb to rice blast disease, appears to have commercial potential.

Using the whole living organism

Bacillus thuringiensis, a bacterium which produces a toxin that kills insects, has the potential of being widely used as a pesticide, though it m a y be far more economical to introduce a gene for the toxin into the plant; this has, in fact, been done for tobacco and tomato. T h e toxin gene of B . thuringiensis has also been introduced into the bacterium Clavibacter xyli which, w h e n m a d e to live in symbiosis with the corn plant, could prevent it from attack by certain pests.

Pseudomonas syringae contains a gene for a protein that acts as a nucleus for formation of ice during frost, leading to frost damage. Genetically engineered organisms devoid of this gene, called ice-minus P. syringae, have undergone field trials in which the damage due to frost was only one-third of that in unprotected plants. A n d Biotechnica International has constructed a strain of Rhizobium that has an enhanced ability to fix nitrogen. Expression in the roots of various plants of genes which synthesize secretory proteins that would solubilize phosphates, might revolutionalize phosphate fertilizer technology.

Scientists have introduced the gene that confers resistance to the herbicide sulphonyl urea into tobacco plants. Monsanto scientists have produced a modified cauliflower plant that produces about twenty times the normal level of 5-enol pyruvyl shikimate-3-phosphate, making it resistant to the herbicide glyphosate. Potato, tobacco and tomato plants have also been rendered resistant to the herbicide phosphinothricin. Similarly, transgenic plants of Brassica napus, one of the most important oil-seed crops of the world, have been obtained that are resistant to methotrexate.

Novel microbial pathways slowly evolve to metabolize environmental chemicals if the selection pressure is low. Using this principle and mixed cultures of microbes, it has been possible to degrade recalcitrant environmental toxin wastes such as dioxins, polychlorinated biphenyls, chlorinated phenols and chlorinated benzenes.

There have been several reports of the development of processes for the bacterial leaching of mineral residues to recover, for example, gold and silver. O n e such report has come from Peru. Certain bacteria, mainly members of the genus Thiobacillus, assist in the leaching operation by converting iron in various compounds from the ferrous to the ferric form. A project is being carried out under the auspices of the U . S . Department of Energy to produce motor fuel from microalgae that exhibit the necessary environmental tolerance, have a high rate of growth and a high lipid content.

O n e more possibility that deserves mention is that of the much-talked-about single-cell protein (SCP), which refers to the dried cells of microorganisms such as algae, actinomycetes, bacteria, yeast, moulds and higher fungi, grown in large-scale culture systems for use as protein sources for h u m a n foods or animal feeds. W e are also likely to use animal and plant cells grown in tissue culture for this purpose in the future.

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Genetic engineering—that is, gene-transfer—techniques, have so far allowed correction of several genetic defects in h u m a n cells and in whole animals. Thus, transfection of cells from a Xeroderma pigmentosum patient with normal h u m a n D N A has been shown to confer resistance to ultra-violet radiation, and it has been possible to correct the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit. Injection of 200 copies of the gene for the basic myelin protein into fresh mouse embryos has been shown to confer resistance to the neurological disease of uncontrollable shivering, convulsions and early death in several strains of mice.

A n d there have been successes with the introduction of genes for proteins with an abundance of essential amino acids, in food crops.

Laboratory synthesis and manipulation

The chemical synthesis of small peptides and proteins has come of age and is n o w commercially viable. S o m e of the proteins and peptides that have been chemically synthesized on a commercially viable scale are calcitonin, secretin, somatostatin, thyrotropin-releasing factor, vasopressin, LH-releasing hormone, glucagon and adrenocorticotropin, all of which have less than 40 amino acids, and seminal plasmin which has 47 amino acids and which has been recently synthesized by D r R . Nagaraj of our laboratory. Japanese scientists have synthesized a peptide that, at a very low concentration, lowers high blood pressure caused by defects in the renin-angiotensin-aldosterone system. Alpha-melanocyte stimulating hormone ( M S H ) which has potential for use in the treatment of melanomas (skin tumours) and other skin diseases and could eventually also have an enormous market as a cosmetic, has also been synthesized. Today, there are many companies around the world which will synthesize a designated gene to order.

There have been reports of a starch-based biodegradable packaging material from the Battelle Institute, London; the Japanese have also developed an opaque, biodegradable plastic sheet using chemical bonding of two or three naturally occurring components derived from plants and microbes.

O n e of the problems of using a modern drug has been that of ensuring that the drug reaches only the target tissue and is released slowly. Encapsulation of drugs within artificial lipid membranes, liposomes, which can be m a d e in such a way that they will go only to the target tissue, is one way of taking care of the above problem. Drugs that have been encapsulated in liposomes and are either already undergoing clinical trials or are likely to do so in the near future, are doxorubicin, platinum L - N D T P , daunorubicin, and a muramyl tripeptide, all for cancer; gentamicin for Gram-negative bacterial infections; amphotericin-B and miconazole for fungal infections; insulin for diabetes; indium for tumour imaging; metaproterenol sulphate for asthma; and minoxidil for hair growth through topical application.

Prospects: near and distant

Ever since the commercial introduction of insulin in 1923, of thyroid hormone in 1934, of factor VIII in 1948, and of calcitonin in 1970, hormones, serum proteins and enzymes have been firmly established as therapeutic agents. M a n y of these proteins have already

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been discussed above. S o m e others that are in use today are: aprotinin from cattle, for stopping bleeding and in the treatment of shock; L-asparaginase from E. coli, for leukemia; collagenase from microorganisms, for healing of wounds; dextranase from the mould Pénicillium, for prophylaxis of caries; hirudin from leeches, for emphysema and prophylaxis of thrombosis; hyaluronidase from cattle, for percutaneous infection; kallikrein from pigs, for circulatory disturbances; lysozyme from chicken eggs, for bacterial infections; thyroid hormones from cattle, for metabolic disturbances; several toxins from snakes and bees, for circulatory disturbances and rheumatism; and several digestive enzymes such as amylase, carboxypeptidase, chymotrypsin, lipase, pepsin and trypsin, from cattle and pigs, and in some cases microorganisms, for digestive disturbances and healing of wounds. S o m e other proteins are angiotensinase from placenta for high blood pressure; and fibrinogen, thrombin, immunoglobulins and plasmin from blood, for blood coagulation diseases, passive immunization and thrombolysis. Proteins of pharmaceutical importance are, today, m a d e by nearly 170 companies in Canada, Japan, the Netherlands, the U S A , the U K , Spain, Brazil, Antilles, Australia, F R G , Denmark , the Republic of Korea, Switzerland, Hungary, Italy, Sweden, France, Argentina and Israel—that is, in at least 18 countries. There is no question whatsoever that these lists will be rapidly enlarged in the decade to come. In the countries named above and in other countries such as India, Ireland and Brazil, there is already substantial investment in biotechnology. In Brazil alone there are at present more than 20 major companies covering a wide field both in terms of research and development and the products they are producing and marketing.

It is estimated that by the end of the century, biotechnology-based chemicals would account for a turnover of over 100 billion dollars per year. It will not be possible to cover all that is on the anvil. Let us, therefore, just take a few examples.

Agricultural biotechnology of tomorrow will include bioprocessing plants engineered to produce pharmaceuticals. For example, today mycobacteria are used to transform the beta-sitosterols 2-androstadienedione and 9-alpha-hydroxyandrostenedione that are important intermediates in the synthesis of steroid hormones. The genes for the enzymes that mediate the above biotransformation can be introduced into plants which would then directly produce the desired steroids. Using genetic engineering techniques and an appropriate probe, sexing of embryos before transfer in embryo-transfer technology, should soon become routine.

Microorganisms can n o w also produce industrial chemicals that can either serve as, or be employed to m a k e , solvents, lubricants, emollients, extractants, adhesives, acidulants, plastics, surface coatings, explosives, propellants, gasoline, additives, alternative fuels, pesticides, dyes, cosmetics, antifreeze, brake fluid, meat tenderizers, digestive aids, vitamins and flavourings. However, out of about 200 substances of commercial value that microorganisms are at present k n o w n to produce, only a few of them are currently m a d e by biological methods in industry; as already mentioned, they include ethanol, n-butanol, acetone, acetic acid, citric acid, lactic acid, amino acids, and enzymes. This number is bound to increase. W a y s will be found for using hydrocarbon-rich wastes of the oil industry for the low-cost production of useful compounds such as amino acids, other organic acids like citrate (used in soft-drink industry), vitamins such as riboflavin and vitamin B 1 2 , sterols such as ergosterol, and certain antibiotics (e.g. of the penicillin-type), making use of genetically engineered microorganisms that would be able to use the carbon-containing substances in the hydrocarbon waste, for growth and conversion into the above-mentioned substances.

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Prospects for the day-after-tomorrow

Let us, again, restrict ourselves to just a few examples. There are already strong arguments in favour of the existence of h u m a n

pheromones—olfactory signals given off by h u m a n beings that m a y be individual-specific (for example, related to or determined by a genetic locus like the major histocompatibility complex that determines the immunological individuality of an individual), and which m a y control or regulate a part of the physiological endocrine response. These signals m a y also regulate the behavioural response of one individual to another, without being registered consciously as olfactory signals. W h e n these signals are identified and their chemistry and physiology understood, m a n would have available a totally new means of controlling and regulating certain aspects of behaviour. The perfume industry m a y then acquire n e w dimensions, and become not only of clinical but also of strategic importance, instead of merely having, quite literally, a cosmetic dimension.

Projects are already under way to transfer the living organism's excellent information-processing functions into electronic devices. These functions include learning, memorizing and pattern recognition. The U K is poised to invest approximately 20 million pounds sterling over a period of five years on the development of molecular electronics.

A n d once w e understand what makes proteins intrinsically unstable, a totally new area will be opened up in designing proteins that will perform the same functions as the originals but with the stability of m a n y everyday inorganic substances.

Benefits and doubts

The benefits of developing and using biotechnology-based products are obvious: their low cost and non-pollutive nature, and the specificity of biological reactions. For developing countries there is an additional advantage of biotechnology being labour-intensive. Therefore, there is no question that more and more chemicals will be produced through biotechnology in the years to come . However, it is not going to be all unadulterated fun.

There will be the problem of contamination with host proteins or other host-derived material which could, for example, be highly immunogenic. N e w ways will, therefore, have to be devised to m a k e sure that h u m a n proteins produced, for example, by E. coli, that are certified for use in h u m a n beings or even animals, are free of E. coli proteins. It follows that clearance of biotechnology-based products from the point of view of their use as food items or as drugs will have to follow a protocol different from those which the foods and drug administrations of various countries are used to. For example, the European Commission has recently banned the use of bovine growth hormone for the fattening of cattle; to m a n y scientists, this would seem to be a decision taken in undue hurry. The same would be true of products that are used in the field, for example for agricultural purposes; the environment protection agencies would need, again, to work out new protocols for their clearance.

The structure of the biotechnology industry will have to be different because of its labour intensiveness—and so would labour-management relations. Then there is the question of patent protection of " n e w " life-forms. There is, perhaps, a case for

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abrogating all patents o n biotechnology-based products and genetically engineered organisms.

Nevertheless, there is n o doubt that w e will learn to cope with the above problems— hopefully sooner than later—and thus m a k e sure that the advantages of biotechnology-based products will far outstrip the disadvantages inherent in this n e w technology. Today , w h e n w e pass a chemical factory, w e very m u c h k n o w that it is there. Perhaps, a time will c o m e soon w h e n w e shall not be able to see or identify a factory from outside, for the chemical industry will have been replaced by biochemical factories, the architecture of which will be fundamentally different. •

Bibliography

(References to the work cited in this article can be found in one or more of the publications listed below.)

A N O N Y M O U S (1989) M o r e wicked ways with hormones. Nature, 338, 281. B A L D W I N , E . , S C H U L T Z , P . G . (1989) Generation of a catalytic antibody by site-directed

mutagenesis. Science, 245, 1104-1106. B H A R G A V A , P . M . (1985) Man and Development, 7 (4), 11-28. B H A R G A V A , P. M . (1989) Social implications of modern biology. India International Quart.,

Spring issue, 173-190. B L O H M , D . , B O L L S C H W E I L E R , C , and H I L L E N , H . (1988) Pharmaceutical proteins. Angew. Chem.

Int. Ed. Engl, 27, 207-225. B R A N G E , J., R I B E L , U . , H A N S E N , J. F . et al. (1988) Monomeric insulins obtained by protein

engineering and their medical implications. Nature, 333, 679-682. C A R T E R , P., N I L S S O N , B . , B U R N I E R , J. P. et al. (1989) Engineering subtilisin B P N for site-specific

proteolysis. Proteins, 6, 240-248. C H A K R A B A R T I , C . (1989) Biotechnology: the basis and the prospects. J. Sei. Industr. Res., 48,

211-228. Genetic engineering and biotechnology monitor (1988, 1989) Nos . 23-26. Vienna, United

Nations Industrial Development Organization ( U N I D O ) . H I A T T , A . (1990) Antibodies produced in plants. Nature, 334, 469-470. K A N E D A , Y . , I W A I , K . , and U C H I D A , T . (1989) Introduction and expression of the h u m a n insulin

gene in adult rat liver. J. Biol. Chem., 264, 12126-12129. K I M , S . - H . , K A N G , C . - H . , K I M , R . et al. (1989) r ^designing a sweet protein: increased stability and

renaturability. Prot. Engg., 2 , 571-576. M A R X , J. (1989) Learning h o w to bottle the immune system. Science, 246, 1250-1251. P O W E L L , M . J. and H A N S E N , D . E . (1989) Catalytic antibodies—a new direction in enzyme design.

Prot. Engg., 3, 69-76. R Ü S S E L , A . J. and F E R S H T , A . R . (1987) Rational modification of enzyme catalysis by engineering

surface charge. Nature, 328, 496-500. S E N O , M . , S A S A D A , R . , I W A N E , M . et al. (1988) Stabilizing basic fibroblast growth factor using

protein engineering. Biochem. Biophys. Res. Commun., 151, 701-708. T A K A N O , T . , N O D A , M . , and T A M U R A , T . - A . (1982) Transfection of cells from a Xeroderma

pigmentosum patient with normal h u m a n D N A confers U V resistance. Nature, 296,269-270. W I D E R A , G . , B U R K L Y , L . C , P I N K E R T , C . A . et al. (1987) Transgenic mice selectively lacking M H C

class II (I-E) antigen expression on B cells: an in vivo approach to investigate la gene function. Cell, 51, 175-187.

W I L S O N , J. M . , J O H N S T O N , D . E . , J E F F E R S O N , D . M . et al. (1988) Correction of the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit. Proc. Natl. Acad. Sei. U.S.A., 85, 4421^1425.

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