Principles of General Physiology

The class of colloidal solutions of most importance to the physiologist is that variously called emulsoid, lyophile, stable, or reversible. These four names, however, although in general applicable to the majority of members of the class, are not, strictly speaking, synonymous. Owing to the existence of all stages of transition, it is natural to find that certain of these characteristics may be absent from a given substance. The word " emulsoid " indicates the liquid nature of the dispersed phase, but, since this phase may contain a greater or less percentage of water, or other solvent, with the same composition of the actual solid matter itself, as especially seen in proteins, it is clear that all degrees may exist between solid and liquid. When the dispersed phase consists of an immiscible liquid, say petroleum, the system exhibits properties approximating to those of the lyophobe class, for example, comparative sensibility to electrolytes (Lewis, 1909, i. p. 493). The fact that very minute drops of liquid have rigidity has already been pointed out, and the further fact that they are retained by the ultra-filter shows that they cannot be sufficiently distorted to be forced through apertures less than of certain dimensions, large in comparison to molecular dimensions.

Again, silicic acid is lyophile, but, after evaporation to dryness, does not again go into solution on addition of water, as gum does. It is then irreversible, contrary to most of the members of the class, which are reversible, in the sense indicated. The designation, "stable," refers to the fact that the sensitiveness to electrolytes is much less than that of the suspended solid particles of the lyophobe class. This, again, is a matter of degree, as facts already given in the previous section (page 92) are sufficient to show. One may also refer to the fact that eggwhite, a typical emulsoid, is precipitated by La--- ion in a concentration of about 0'002 molar, whereas arsenious sulphide, as we have seen, reacts to the same ion in 0-00005 molar. Remembering the ratio of activity of ions of different valency, it is not surprising that univalent ions are practically inactive on emulsoid colloids, that is, so far as their effect as charged ions is concerned.

Mines (1912, p. 211) has found a useful test for the emulsoid state. This consists in the reaction to complex trivalent ions as compared with that to simple trivalent ions. Cobalt, and some other metals, form complex salts with ammonia and an acid ; these are electrolytically dissociated with the formation of a large trivalent cation, such as Co(NH3)g---(luteo-cobalt) ion ; emulsoids, such as eggwhite, are not precipitated, even by comparatively high concentrations of this ion, up to 0'02 molar, whereas suspensoids are nearly as sensitive to it as to the simple, La-", ion.

Egg-white, coagulated by boiling, behaved in this respect as a suspensoid. As Mines points out, tea infusion contains suspensoid colloids, whereas cream is an emulsoid , so that opportunity for testing the different behaviour is ready to hand. Silicic acid seems to be an exception ; although showing most of the characters of emulsoids, it is as sensitive to the complex trivalent ion as to the simple one. A fine emulsion of olive oil behaves as an emulsoid to trivalent ions.

The facts of the preceding paragraph show that valency is not the only factor concerned in the action of electrolytes, even in that aspect of their action connected with the electrical charge. There are two ways in which the complex ion differs from the simple one, viz., its slow rate of movement, and the less density of the charge on its greater surface. Mines (1912, p. 235) calculates the relative density on the lanthanum and luteo-cobalt ions as being in the ratio of 1'37 to 0-26, and suggests that the power of adhesion to the particle to be discharged is in relation to this fact.

The two phases of which hydrophile colloids consist differ only in the relative amount of water and solid in each. It will readily be seen, therefore, how the properties can be altered by agencies capable of changing this distribution of water. This point has been especially insisted on by Hatschek (1913, p. 46). If the water content of the internal phase is diminished far enough, this phase will become solid, and the system will be a suspensoid one. With large water content of the internal phase, its properties will approach to those of a liquid, and the system will be an emulsoid one. The "salting out" of proteins, etc., by high concentiations of electrolytes is due to removal of water from the internal phase, and consequent precipitation of this latter. The way in which water is present in emulsoids is regarded by Hatschek as similar to imbibition, which will be discussed later, although the possibility must not be lost sight of that more strictly chemical affinities may play a part.

When we consider the series of salts investigated by Hofmeister (1888), as regards their relative effect on the salting out of albumin, and known as the " Hofmeister series" no obvious reason is apparent for the different behaviour of the salts. A chemical one is excluded by the fact that the same series is found in the action on substances so different in constitution as albumin, gelatine, agar, and starch. As Hatschek (1912, p. 46) points out, the only view which co-ordinates the various phenomena is that they are all manifestations of a change in the distribution of water between the two phases ; the salts of the Hofmeister series do this by their action on the compressibility of water. The solution of emulsoids is usually associated with contraction. These phenomena, in general, belong "to that class called by Freundlich " lyotropic " (1909, pp. 54 and 412), as dependent on changes in the solvent itself. When water is the solvent, we speak of the hydration of the ions of the salt, and the changes in the equilibrium between the various molecular states of fluid water. These changes give rise, in their turn, to alterations in the internal pressure, expressed in changes of compressibility, viscosity, solubility, and so on. For further details as to the Hofmeister series, and the action of salts on emulsoids, the reader is referred to the book of Freundlich (1909, pp. 424 seq.).

It was known to Faraday (1858, p. 175) that the precipitating action of "salt " on gold solutions could be prevented by the addition of a trace of "jelly." Other emulsoid colloids have this action, although in different degree, and the fact serves as the basis for the "gold number " of Schulz and Zsigmondy (1903) as a characteristic of individual proteins. It seems certain that this protection against the action of electrolytes conferred by an emulsoid on a suspensoid is due to the deposition of a film of the former over the surface of the solid particles, thus practically converting the system into an emulsoid one. Mines (1912, p. 219), by the application of his test with complex trivalent ions, finds that a gold hydrosol protected by an emulsoid behaves in the same insensitive way as the emulsoid itself. Moreover, if the protective colloid be a protein, which has the sign of its charge easily reversed by acid, as gelatine, it will be found that the gold particles, previously insensitive to acid, have become sensitive (ibid., p. 222). It can be shown by electric convection that it is not easy to reverse the sign of the charge on gold particles by acid alone ; when they are coated with gelatine this is easy, although gelatine itself does not affect the sign of the charge. The adsorption of protective colloid by the surface of the gold particles is no doubt due to the lowering of surface tension thereby brought about ; and the gold number varies according to the capacity in this respect.

The protective action is not necessarily complete, as I noticed in some experiments made with arsenious sulphide and with Congo-red. In these cases, actual precipitation by calcium sulphate was prevented by the addition of albumin, as in the case of gold, but if such mixtures were carefully compared with the original, it was noticed that they were somewhat more turbid. Under the ultra-microscope, the change was very obvious in the case of Congored. This dye, in the absence of electrolytes, is not resolvable into particles. After the addition of serum-albumin and calcium sulphate, although no precipitation occurred, as when the salt was added alone, the solution was nevertheless found to be full of very distinct, but not brilliant, particles. This effect is in agreement with the small, but not negligible, effect of salts on emulsoids.

Walpole (1913, 3) has shown that gelatine in very low concentration (1 in 100,000,000) increases the effect of hyfoochloric acid in the aggregation of hydrosols of gold, mastic or oil. In concentrations of 10~6 to 10~4'4 of gelatine there are two concentrations of the acid which produce aggregation, whereas, between these two, no effect is produced. In cases of aggregation due to the assistance of a "protective " colloid, reversal is obtained by the addition of alkali, and ultra-microscopic examination shows that the aggregates in the case of oil solutions consist of numbers of the original minute particles, stuck together ; whereas, in the case of aggregation by hydrochloric acid in the presence of gelatine of concentration lower than 10~8, the aggregates are comparatively large drops of oil. There is no change of sign of the electric charge in these cases of aggregation brought about by traces of gelatine. When concentrations of gelatine greater than 10~4-4 protect from the action of acid, the sign of the charge of the particles is converted from negative to positive by the acid. For further details see Walpole's second paper (1914, 2).

A marked difference in viscosity, or internal friction, is shown by emulsoids and suspensoids. While that of the latter is not greatly different from that of water, the former, as a rule, have a very considerable viscosity (Freundlich, 1 909, p. 396). The various ways in which this manifests itself are only to be explained by the assumption that we have to deal with a diphasic system of two liquid phases (see Hatschek, 1913, p. 43). In contact with a solid surface, drops of the more tenacious phase adhere, and, as the fluid is forced past, these drops are deformed and torn apart. Another interesting fact, which proves the origin of the high viscosity in a two-phase nature of the system, is that mechanical deformation produces double refraction (Kundt, 1881, p. 110). In homogeneous viscous liquids, such as glycerol, strong sugar solutions, etc., this is not to be detected, whereas in gelatine, even of O'Ol per cent., double refraction can be produced by mechanical stress, which places the dispersed phase in a state of asymmetrical tension.

The viscosity of emulsoids has a high temperature coefficient. There are two conditions very frequently met with in emulsoids, the phenomena of gelatinisation and those due to imbibition of water or other solvent. The following two sections deal briefly with these. When a gelatine solution, which is a freely-flowing liquid at temperatures above 20°-25°, is cooled, it " sets " to a substance having the property of preserving the shape into which it is trimmed. It has, also, elasticity of form, so that, within limits, it returns to its original form after distortion. What has taken place ? In speaking of the action of fixing solutions on protoplasm, the experiments of Hardy (1900, 2) were referred to. This investigator showed that gelatine, when simply cooled and unacted on by reagents, required an enormous pressure to squeeze out any of the water which it contained. This fact means that the water no longer forms a continuous phase, but must be enclosed in vesicles composed of the more solid phase, so that, to escape, the water must pass through gelatine. Fig. 15, B (p. 14), represents diagrammatically the state of affairs, if we regard the black as gelatine (containing water), and the white the liquid phase, that is, dilute solution of gelatine. From what we have learnt above as to the nature of emulsoids, it is clear that the word " solid " phase, used in describing the phenomena, must be understood as " relatively more solid " phase.

From Hardy's work (1900, 2) it appears that the first sign of commencing gelation is a change of the system from a micro-heterogeneous one to a more coarsely heterogeneous one, so that drops of the dispersed phase separate. It is interesting to note the proof afforded by this fact of the liquid nature of the internal phase of an emulsoid, since only a liquid could form drops. The further fate of the drops depends on the concentration of the solution. In very dilute solution, the droplets remain sufficiently small to become a permanent dispersed phase, freely movable and, in fact, showing Brownian movement. When the solution is more concentrated, the droplets join together to form a network or similar kind of structure, but the watery phase is still continuous. When still more concentrated, the droplets which separate can be seen by their refraction to consist of the watery phase, so that the more solid phase has now become the continuous or external one, while the more liquid one is the internal or dispersed phase. The change described above is a reversible one, and is important as illustrating the kind of phenomena which may occur isothermally in a complex system of emulsoids, such as living protoplasm.

The composition of the two phases of a colloidal system, as not being qualitatively, but merely quantitatively, different, is well seen in the following figures (Hardy, 1900, 2, p. 257) from a case of a ternary mixture of gelatine, alcohol, and water. The numbers represent grams of gelatine per 100 c.c. of the gelatine solution at 15°. The work of Bachmann (1912) and of Zsigmondy (1913) on the formation of gels, as observed with the ultra-microscope, is of interest. Most of the work was done with pure soaps and is illustrated by Fig. 43. It is well known that a fairly strong hot solution of sodium or potassium stearate or palmitate sets to a more or less transparent, tenacious jelly, when it cools. This usually changes later into an opaque, white, friable mass. The former corresponds to a fine felt-work, as seen under the ultra-microscope (D and F of the figure) ; while the latter is obviously crystalline (D in the figure). The structure of the gel, as first formed, shows a strongly polarised cone of light and is, therefore, of an extremely fine degree of heterogeneity, much finer than the foam structures described by Blitschli. As cooling proceeds, the particles become larger, Brownian movement is easily seen (A). These particles continue to increase in number, obstruct one another in movements, and suddenly form threads, which are said to have a "crystalline"

FIG. 43. DIAGRAMS OF ULTRA-MICROSCOPIC APPEARANCE OF SOAP GELS IN PROCESS A, B, C, D, E, Stages of gelation and crystallisation of 5 per cent, potassium stearate in water. Enlarged about 200-300 times. F, Jelly of 10 per cent, sodium oleate. Felt-work obtained by dissolving away the finer threads. Cardioid G, Jelly of 5 per cent, sodium palmitate. Needle-like fibres. H, Crystals of potassium stearate from watery solution of about 10 per cent. Final state. Magnified about 300 times.

appearance (see F and G), and result in the production of a feltwork. After a time, this felt-work changes into distinct separate crystals (E and H). Whether the first particles are to be regarded as " micellae," in Niigeli's sense, that is, as aggregates with crystalline properties, is a matter for argument. It is clear, however, that the vectorial forces, which ultimately result in the formation of distinct crystals, must be always present, but apparently require time for action. The ultra-microscopic particles, probably of a crystalline form, at first separate out arranged in threads and networks.

An important point in regard to the nature of the two phases is that Bachmann found that the " inter-micellar " fluid, in the case of a gel of 1 per cent, sodium palmitate, contained 0-06 per cent, of the salt. Many emulsoids, after being dried, are capable of taking up again large quantities of water, without actually forming liquid solutions, such as ordinary hygroscopic substances, calcium chloride, for, example, do. Most parts of plants and animals exhibit this property to a greater or less degree. The stalk of the sea-weed, Laminaria, increases enormously in volume under the conditions mentioned and has been made use of in surgical practice.

The greater part of the experimental work on imbibition has been done on gelatine, a considerable amount also on starch. Perhaps the most striking thing about the phenomenon is the great pressure exerted in the process of swelling, or conversely, required to express water after it has been taken up. Laminaria, under a pressure of 42 atmospheres, was found by Reinke (1879) to be able still to take up 16 per cent, of water. In all these processes, it is important to remember that the total volume, gel plus water, is less after swelling, although the volume of the gel itself increases so much. In order to compress water to the extent implied in the total change of volume, a pressure of some 300 atmospheres is necessary, so that it is plain that heat must be evolved in the process of imbibition.

This compression of water can be demonstrated in the following way, due to R. du Bois- Reymond (1913). Pieces of the dried material, such as Laminaria, are attached to the submerged part of a hydrometer, and the scale adjusted to a convenient point by addition of weights. As the material swells, the hydrometer sinks, showing that the water which has become part of the imbibition system has increased in density. Of course, the temperature must be kept constant.

Much work has been done on the effect of electrolytes on the swelling of emulsoids, especially of proteins. The most striking effect is that of acid and of alkali. Spiro (1904, p. 276) showed that either of these greatly increases the amount of water taken up by gelatine, and Chiari (1911) found that, when carefully purified, gelatine is sensitive to very small differences in H- ion concentration, so that the difference between ordinary distilled water and that distilled out of contact with carbon dioxide may be detected. The explanation of this phenomenon, as given by Pauli (1912, p. 262), is that electrolytically dissociated salts of protein are formed by acid and by alkali, and the swelling is due to the atlinity for water of the protein ion. This view will be discussed under the head of proteins.

A theory of (edema has been propounded by Martin Fischer (1910) on the basis of the action of acids on the swelling of proteins. The tissue colloids are supposed to take up water under the influence of increased acid reaction of the blood. Although the possibility of such effects must not be forgotten, they will not easily explain the actual presence of liquid in dropsical tissues ; a fine canula or hollow needle inserted into such tissues allows a slow stream of fluid to drop from the end, and it is well known that oedema passes from one part of the Ixxly to another in obedience to gravity. Moreover, so far as I am aware, M. Fischer has not actually shown a change in FT ion concentration in the blood sufficiently large to account for the effect. As we shall see in Chapter VII., the chemical composition of blood is such as to form an extremely efficient arrangement for keeping the reaction constant. And again, this delicate sensibility to change of H* ion concentration shown by gelatine is only manifested in the absence of neutral salts, a condition not met with in living organisms.

The work of Siebeck (1912, p. 467) on kidney cells, and of Beutner (1913, p. 224) on muscle, lead them to the conclusion that the increase of size, occurring in certain solutions, is due to osmotic taking up of water, rather than to an imbibition process, and that acid or alkaline reaction has no effect unless the cells are permanently injured. Moreover, the action of neutral salts on the volume of cells is in proportion to their molecular concentration only, whereas the effect on imbibition is different according to the chemical nature of the salt, even when in equi molecular concentrations.

Hofmeister (1888), in fact, found the action of neutral salt* on the process of imbibition to follow the same series as that already mentioned in the case of "salting out." The relation of this series to the properties of the solvent has been indicated on page 97 above. Samec (1911, p. 156) calls attention to the fact that, parallel to the favouring effect exerted by the anions of the Hofmeister series on the imbibition of water by starch, there runs a set of

physico-chemical properties of the salt solutions themselves. These are, rate of diffusion and compressibility, which increase with the favouring action, while surface tension, internal friction, electrical conductivity, diminution of solubility of other solutes, maximum density, effect on catalysis of esters, inversion of cane-sugar by acids, and dissociation of weak acids are properties which decrease along with increase of favouring action. At first sight it would seem natural to connect these various phenomena with hydration of the respective crystalloids in solution. A part of the solvent is held in this way in the region of the solute, so that any process in which water is concerned would pursue a different course in presence of crystalloids than in their absence, and, in general, the change would be of the same kind as that caused by increase in concentration. There seems, however, to be some additional factor, because there are some crystalloids whose solutions produce wore swelling than pure water does. The suggestion is made by Samec (p. 157) that adsorption of the crystalloid takes place on the surface of the gel elements and that the adsorbed, highly hydrated, substance brings water into more intimate contact with the colloid. The fact that any particular ion has precisely the same effect on a protein and on starch, as pointed out by Samec (1911, p. 154), shows that the formation of chemical compounds does not play any important part. Further information as to the behaviour of gelatine in various solutions in water, may be found in the paper by Ehrenberg (1913).

As to the nature of the process itself, Posnyak (1912, p. 154) calls attention to three possibilities : — 1. Condensation of water on the surface of the elementary particles of the gel, leading to filling up of the capillary spaces between them, while the particles themselves remain unchanged in size. 2. Simple solution of the liquid in the substance of the particles, which thereby change their size, density, etc. 3. Both processes take place. This is regarded by Posnyak as the most probable one theoretically, although his experiments on the influence of pressure on the liquid content of a gel speak more in favour of the first. He finds, in fact, that the content in solid (c) of such gels as india-rubber and gelatine is related to the pressure (P) by the formula : —

where A and k are constants. A varies considerably from gel to gel and from liquid to liquid, while k has always the same value ( = 3). This latter fact is difficult to explain on the basis of a solution of the liquid in the colloid substance and consequent change in its properties. According to Zsigmondy (1913) the lowering of vapour pressure in the imbibition of water by silica gels is due to the formation of a concave meniscus, not to formation of hydrates. Imbibition is the filling of hollow spaces in this case, not the taking up of water into actual substance.

The similarity of Posnyak's equation to the simple form of the expression for adsorption is obvious. It would seem also to be more advantageous for rapid changes in the distribution of water, such as are required in physiological activities, that the water should be on the surface rather than inside the substance of the colloid. As Posnyak suggests, it is probable that the relative share taken by the two kinds of process differs according to the amount of water available.

Some experiments which I have recently had occasion to make favour this suggestion. Gelatine is sometimes used to remove water from alcohol that is nearly absolute, say 90 to 95 per cent. Of course, it is useless for this purpose unless thoroughly dried first. I found that it does remove water from 90 per cent, alcohol, so that this becomes stronger, but, to my surprise, no increase in volume of the gelatine was to be detected, although the amount of water removed from the alcohol was sufficient to be detected easily. The gelatine also appeared to be just as hard and horny as when put in. The only explanation seems to be that, in order to determine the volume after immersion in alcohol, the pieces were allowed to dry for about a minute in air ; the liquid alcohol evaporated from the surface in this process, and apparently the water concentrated on the surface passed off with the alcohol, a phenomenon that could not have taken place in so short a time if water had penetrated into the substance of the gelatine.

The facts above described will be found in later pages to have a bearing on the action of enzymes. In many respects proteins are the most important members of the emulsoid class and, at the same time, the most difficult to treat in a satisfactory way. This difficulty is mainly due to the fact that the phenomena presented by them can be, for the most part, described from two different points of view, from that of pure structural chemistry and from the physico-chemical standpoint of colloidal chemistry.

Take, for example, the common test for the presence of a protein in solution, that with potassium ferrocyanide and acetic acid. Potassium ferrocyanide alone, in low concentration, does not give a precipitate, hut such appears when the solution is ni;u It- acid with acetic acid. This may be explained by saying that the compound of protein with ferrocj'anide is soluble in neutral or alkaline solution, insoluble in acid. Or by saying that the negative ferrocyanic ion has no precipitating action on an electro-negative colloid, as protein is in neutral or alkaline solution, but becomes a powerful one, as a quadrivalent ion, when the colloid is made positive by the H' ion of an acid.