Principles of General Physiology

To neutralise ricin, the toxic substance from the castor bean, it was found that less antiricin was necessary if addtd to a definite amount in successive quantities than if added all at once. And, if ricin be added in separate doses to a definite amount of antiricin, the same amount of ricin requires more antiricin to neutralise it than if the whole is added at one time. This phenomenon also takes place when paper adsorbs Congo red (Bayliss, 1906, p. 222). The explanation is that the same amount of adsorbent will take up relatively more from a dilute solution than from a more concentrated one.

5. In the taking up of bacilli by leucocytes under the influence of a sensitising fluid (so-called "opsonin"), it was found by Ledingham (1912, p. 359) that the two processes involved both followed the course of an adsorption process. These two parts of the phenomenon are (1) the taking up of " opsonin " by the bacilli, and (2) the ingestion by the leucocytes of the micro-organisms thus "sensitised." 6. When the toxin of tetanus is introduced into a nerve trunk of a warmblooded animal, it is carried up to the central nervous system and produces convulsions in. due course. If the same experiment be performed on a frog at 8° C. it was found by Morgenroth (1900) that although taken up by the nervous system, no convulsions were produced until the animal was warmed to a temperature of about 20° C. This is evidently a similar case to that of the Congored acid and thorium hydroxide described above. The toxin, although adsorbed, exerts no action until chemical reaction of some kind takes place on warming.

7. The rate of action of enzymes is controlled by adsorption, but full discussion will be more conveniently deferred until later. 8. When the protoplasmic contents of a ciliate infusorian or the root hair of a plant are pressed out into water, a membrane is at once formed on the free surface of the protoplasm. This fact has been described by Kuhne (1864, p. 39) and by Pfeffer (1897, i. pp. 92, 93). The nature of this membrane will be discussed in Chapter V., and it will suffice to call attention to it here as being undoubtedly due to surface concentration of cell-constituents which lower surface energy.

9. The blue substance formed by the action of iodine on starch has long been familiar, but its nature as an adsorption compound has only recently (1912) been made clear by the work of Barger and Field (1912). They also show that similar blue compounds are formed by substances of very varied chemical nature, such as saponarin, cholalic acid, and lanthanum acetate. 10. That powerful action on cell processes can be exerted by substances which do not penetrate beyond the surface of the cell is shown by a very interesting experiment of Warburg (1910, pp. 310, 311, 313). The oxygen consumption of the fertilised eggs of a sea-urchin in an artificial sea-water is doubled by the addition of 10 c.c. of decinormal sodium hydroxide to 1 litre of the sea-water, the development being, at the same time, stopped. If the cells are stained previously with neutral red, which does not affect their development, no change of colour takes place on addition of sodium hydroxide ; whereas with ammonia, to which the cell membrane is permeable, the cells become yellow in less than one minute. Athough uninjured by the concentration of ammonia used, the oxygen consumption is only increased by 10 per cent, instead of the 100 per cent, when the H* ion concentration is changed only at the surface.

There is one point that it is of some importance to understand clearly. When an electrolyte, say acetic acid, is adsorbed by charcoal, it is fixed for the time on the surface. By this statement it is not meant to imply that the same identical molecules remain in the same place, but that a certain proportion of the acid is taken out of solution and cannot take part in such properties as the electrical conductivity or the osmotic pressure of the system. A mixture of acetic acid and charcoal has the electrical conductivity of the liquid phase alone. Similarly, when the particles of the adsorbent are too large to give an osmotic pressure (see Chapter V.), the osmotic pressure of the system is due only to the solution. The adsorption compound of charcoal plus acetic acid, or other adsorbed electrolyte, has no higher osmotic pressure nor conductivity than the charcoal itself. Some observers are inclined to attribute, incorrectly, the osmotic pressure undoubtedly shown by certain colloidal solutions to adsorbed electrolytes or crystalloids.

In the state of adsorption, salts, not being electrolytically dissociated, give none of their characteristic reactions. Iron, for example, is in what is sometimes called a "masked" condition. Ruer (1905) found that when chlorides are adsorbed by colloidal zirconium hydroxide, no reaction with silver nitrate is given. The presence of chlorine in colloidal ferric hydroxide can only be detected by transforming the colloidal solution into a true solution by means of nitric acid, that is, by abolition of the adsorbing surface.

On the other hand, it must not be forgotten that adsorbed substances are only fixed as long as the solution with which they are in equilibrium remains of the same concentration, which may, however, be very low. Nevertheless, by repeated washing, practically the whole of the adsorbed matter may be removed, although an infinite number of changes of water is theoretically necessary. If charcoal which has adsorbed sugar be placed inside an osmometer, whose membrane is permeable to water and sugar, but not to charcoal, sugar will pass out to water on the outside, and by repeated changes of this water the sugar can be almost entirely removed from the charcoal inside. Substances merely adsorbed cannot be prevented from escape to water ; in order that they shall not do so, they must

be in a state of non-dissociable chemical combination with the substance to which the membrane is impermeable. If a surface which has adsorbed a particular substance be exposed to a solution of another one which has a greater power of lowering surface energy than the first, there is a more or less complete displacement of the less powerful one by the other. This is shown in an interesting way in the experiments of Schmidt-Xielsen (1910, p. :U2). When rennet is shaken up in solution, it is more or less inactivated by adsorption on tho surface of the froth produced. This inactivation is completely absent if a little saponin be added, although the foam is even greater than before. Saponin, in fact, lowers surface energy more than does rennet, hence it obtains possession of the surface. The same fact is seen in the driving out of rennet from its adsorption by charcoal in the experiments of Jahnson-Blohm (1912). Charcoal added to rennet prevents its action on milk (acting as an anti-enzyme), but, if saponin be added to such an inactive mixture, it becomes active owing to the driving off by the saponin of the rennet from its " combination with the antibody."

This fact, that one substance can displace another from adsorption, is of importance with respect to the turning out of oxygen from oxvhsemoglobin by exposure to carbon monoxide (see Chapter XXL). A short account may be given here of the bearing that the facts of the present chapter have on the nature of the processes involved in the dyeing of fabrics, and in the similar art of staining histological preparations, as no further opportunity will present itself.

Much controversy has taken place between advocates of chemical and physical theories. It may be taken as established that a physical theory, based only on coefficients of partition, due to greater solubility of dyes in the tissues than in the staining solution, is inadequate. On the other hand, many facts have been mentioned in the preceding pages which indicate the important part that surface action, or adsorption, must play, as well as the probability that chemical reaction may, in many cases, follow it, although adsorption is the controlling factor.

Weber (1894) finds that the amount of dye taken up by cellulose is in proportion to the extent of surface presented by the latter. Precipitated cellulose takes up more than does an equal weight of compressed paper. Dinitrocellulose, freshly precipitated, adsorbs in about the same degree as ordinary cellulose ; but in the form of a coherent film little or none is taken up. In discussions on the subject of staining, the use of the names " basic " and " acidic " is liable to lead to some misconception. With one or two exceptions, all dyes are neutral salts ; the distinction is that the so called " basic " dyes are salts of an organic coloured base with an inorganic acid, usually h3Tdrochloric, although sometimes salts with acetic acid are met with. The "acid" dyes, on the other hand, are salts of a coloured organic acid with an inorganic base, usually sodium.

Bearing this fact in mind, it is clear that, if a " basic " dye stains a particular cell constituent, it does not directly follow that this constituent is an acid. If such, it must be a stronger acid than that combined with the colour base of the dye, usually hydrochloric. Double decomposition may occur, of course, if the cell constituent in question is a salt. This will be more complete the less soluble the compound between dye and tissue is. Similar statements apply, mutatis mutandis, to " acidic " dyes. Since most of the staining bodies in cells are colloids and with negative charges, it is easy to understand why electropositive dyes, such as many of the " basic " ones are, should be adsorbed. It is also suggestive that haemoglobin, one of the few electro-positive colloids of the organism (Iscovesco, 1906), takes up "acid" dyes, such as eosin and acid fuchsin. Moreover, when the dye salts are electrolytically dissociated, as in most cases, the positive ion is the coloured one in the "basic" dyes, and will be taken up by negative surfaces, while the negative ion of the "acid" dyes will be taken up by the positive surfaces. The " basic " dyes are frequently hydrolytically dissociated, with formation of electro-positive free bases in the colloidal state.

There are some more facts of interest, tending to show the great importance of the electrical charge of the surface. Gee and Harrison (see William Harrison, 1911, p. 6 of reprint) found that the maximum negative charge of cotton, wool, and silk was at a temperature of 40° C. Brown (1901, p. 92) had previously shown that the maximum adsorption of "basic" (electro-positive) dyes by wool took place at the same temperature. W. Harrison (1911, p. 26) also showed that cotton treated in various ways, nitrated, mercerised, and so on, had a contact potential difference against dilute sodium chloride whick differed considerably in amount according to the treatment, although the charge was always negative. The amount of "acid" ( = electro-negative) dye adsorbed was parallel with the decrease, in the charge.

That the deposition of an electro-positive dye on a negative surface results in a lowering of the charge on this surface is shown by an experiment of Larguier des Bancels (1909). The charge on wool, as measured by the number of drops of water transferred from one electrode to the other, in an apparatus similar to that of Perrin (1904, p. 616), in a given time was represented by 77. After staining with methylene blue, the number was reduced to 18.

The very marked effect of electrolytes in altering the charge on surfaces has been frequently referred to, as also the fact that electro-negative substances are scarcely adsorbed at all by electro-negative surfaces. In order that this adsorption may take place, the surface must be discharged, or the amount of its charge lessened, by the action of an ion of opposite sign. That very small quantities of an appropriate ion suffice is well shown by an experiment of Elissafoff (1912, p. 404), whose work will be referred to in more detail in the chapter on " The Colloidal State." 0-2 mgm. of thorium nitrate per litre lowered the charge on the surface of a quartz capillary by 50 per cent.

The absence of staining by " acid " dyes in the absence of electrolytes explains why fresh teased nerve fibres of the frog only stain with Congo-red at their cut ends, where, according to Macdonald (1905, p. 329), electrolytes are set free. Emil Mayr (1906, p. 560) finds that the affinity of Nissl bodies in nerve cells for " basic " dyes is reduced by previous treatment with neutral salts. This fact also is in agreement with the doctrine of electrical adsorption. The Nissl bodies have, in all probability, a negative charge; this charge would be diminished by cations, and hence the attraction for positive substances, like the " basic " dyes, would be also diminished.

Disregard of this action of electrolytes has led to certain erroneous statements with regard to dyes. It is to be remembered that commercial specimens almost invariably contain a large percentage of salts, frequently as much as 20 to 30 per cent, of sodium chloride or sulphate, arising from the mode of preparation. When it is said that Congo-red is a " direct " dye for cotton, the statement only applies to the commercial dye, with its content of salts. When adsorption, moreover, takes place under the influence of electrolytes, it is, as a rule, " faster," that is, not so easily removed by the action of water, than when it takes place in their absence. This applies more especially to the electro-negative dyes.

Certain facts described as " anomalous adsorption " (Biltz, 1910) will also be found to be explained by the presence of electrolytes (Bayliss, 1911, 3). For many purposes it is necessary to have pure dyes. The following method, due to Harrison (1911, p. 17), may be recommended. It depends on the displacement of the nonvolatile salts, present as impurity, by a volatile one. The dye in concentrated solution is precipitated by saturation with ammonium carbonate ("salted out"), redissolved in water, and again salted out. After washing with a saturated solution of ammonium carbonate, the precipitate is dried at 110° C., when all the ammonium carbonate is driven off.

A curious fact was noted by Freundlich and Losev (1907, pp. 311, 312) : When an " acid " dye is adsorbed, the whole of the molecule is taken up. When a "basic" dye is adsorbed, the positive coloured ion only is taken up, leaving the acid. Satisfactory explanation of these facts is not at present at hand (see Freundlich and Neumann, 1909). There are, however, two facts to be remembered in this connection. The "acid" dyes are, as a rule, sodium salts of strong (sulphonic) acids and are very little, if at all, hydrolysed in solution, but electrolytically dissociated to a considerable degree. The anion, containing a large number of atoms, seems to behave as a colloid and has, of course, a negative charge. The " basic " dyes, on the other hand, are salts of a rather feeble organic colour base with a strong acid and are hydrolysed in solution. The free base is insoluble, in the ordinary sense, but forms a colloidal solution, the particles having a positive charge. The behaviour of the two classes may perhaps depend in some way on this difference in mode of dissociation.

The surface of contact between a liquid and another phase — solid, immiscible liquid, or gas — has properties differing from those of the main body of either phase. In the first place, the surface film behaves as if stretched, so that it is the seat of a special kind of energy. This surface tension has its origin in the forces of attraction between the molecules of the liquid, the forces which give rise to the internal pressure of Laplace. The amount of this surface energy varies with the chemical nature of the liquid.

All solutes, with the exception of certain inorganic salts, lower the surface tension at the interface between liquid and air; these particular salts do so at the interface between liquids. The interface between phases is also nearly always the seat of electrical forces, the origin of which is usually from electrolytic dissociation in one or other of the phases. But the possibility of phenomena akin to those of frictional electricity cannot as yet be definitely excluded.

Since any process that diminishes free energy tends to occur, a solute will bo found in higher concentration in the surface film than in the body of the liquid if it has the power of reducing surface energy. By this means, a greater fall in surface energy is ensured. (Principle of Willard Gibbs.) This surface condensation is known as " adsorption " and plays an important part in physiological phenomena. The surface energy spoken of in the previous statement of the Gibbs principle may be of many kinds, mechanical, electrical, chemical, etc.

In certain cases, surface concentration leads to the formation of a more or less rigid film, as, for example, with saponin or proteins (Ramsden). When a solute has an electrical charge, either as an ion or as a colloidal particle, and the surface in contact with the solution has also a charge, the degree of adsorption depends on the relative sign of the two charges ; no decrease of free energy would be produced by adsorption of a negatively charged substance on a similarly charged surface, but the reverse. On the other hand, adsorption of an oppositely charged substance leads, by neutralisation of the charge, to decrease of free energy.

If the surface has no charge, while the adsorption of an electrically charged ion would lead to diminution of mechanical surface energy, such adsorption will take place and cause the appearance of an electrical charge on the surface. Adsorption of a similarly charged ion or colloid can be increased by reversing the sign of the charge on the surface by allowing it previously to adsorb ions of sign opposite to itself. If the surface and the solute have already opposite signs, it is clear that previous adsorption by the surface of an opposite charge will decrease subsequent adsorption of the particular solute.

These phenomena of electrical adsorption play a considerable part in the processes of dyeing and of histological staining. Chemical reactions which lower chemical potential are also favoured at a surface. The rate of such reactions is not controlled by the total mass of the reagents, as in true solution, but by the extent of active surface. The law of mass action, in its simple form, does not apply quantitatively, since the surface of one or both of the reagents has to be taken into account.

There is some evidence that the chemical configuration of the surface may play a part in adsorption and lead to the appearance of "specific" action. But the question needs further investigation. The rate at which adsorption takes place, when the components are already approximated, appears to be very rapid, although not instantaneous. On the other hand, when the substance to be adsorbed has to diffuse from distant parts of the system, the rate will be controlled by diffusion and therefore accelerated by rise of temperature.

The total amount adsorbed in equilibrium is less the higher the temperature. The process, by the "principle of mobile equilibrium," is, therefore, associated with the production of heat. The mathematical form of the expression relating concentration with amount adsorbed is a characteristic one and belongs to the parabolic family. Adsorption cannot be completely or satisfactorily explained by chemical combination, nor by partition between phases, in accordance with relative solubility, since impossible assumptions have to be made as regards molecular association, etc.

A class of compounds exists in which, as shown by various facts, the constituents are not chemically combined. This is shown especially by the dependence of their composition on the relative concentration of the substances from which they are produced. This class of compounds is satisfactorily explained by adsorption. In some of these adsorption-compounds the constituents can be shown to be present, side by side, but uncombined. Since the rate of chemical action depends on the concentration of the reagents it is plain that when a substance is capable of reacting with a second one, which is present as a separate phase, particles or drops, for example, the rate of reaction will depend on the amount of the one adsorbed on the surface of the second. Similarly, if two substances, capable of reacting with each other, are both adsorbed on the surface of a third, with which they do not combine, their rate of reaction with each other will be accelerated by the increased concentration, or molecular approximation, due to adsorption.

A number of cases are given where adsorption plays a controlling part in phenomena of physiological interest. Salts when adsorbed are not electrolytically dissociated and do not therefore give their characteristic reactions, neither can they be osmotically active. A substance which lowers surface energy more than a second one does will drive off this latter from adsorption on a surface, at the same time taking its place thereon. Adsorption plays a large part in the phenomena of dyeing and staining ; most, if not all, the facts can be explained on this basis ; although, in all probability, chemical reaction sometimes follows adsorption, the rate of this reaction being controlled by the amount adsorbed.

Adsorption preliminary to Chemical Action, Baylies (1911, 1). IF we take a piece of metallic gold, immerse it in water, and divide it up into smaller and smaller parts, it is obvious that in the end, supposing that our powers of manipulation were adequate, we should arrive at the molecular condition. But, before this state is reached, we should have passed through a state in which the particles were so fine as to be invisible, as such, by ordinary means of illumination ; and they would remain in permanent suspension, so as to simulate very closely a true solution, in which the substance dissolved is in the molecular, or even ionic state. In the course of this process of division, the larger fragments of gold of the early stages sink at once, after being stirred up, but as smaller and smaller particles are formed, the time taken to fall becomes longer and longer, until, when less than a certain size, they do not appear to sink at all. They are now in what is called the " colloidal state." Their dimensions at this stage are enormously greater than those of molecules of gold, but it is clear that we can draw no definite lines of demarcation between the visible solid lump, from which we started, the colloidal state and the final molecular state.