Principles of General Physiology

It is indeed sometimes a difficulty in analytical work that precipitates will not deposit because of the absence of electrolytes to cause their aggregation. Sometimes it is possible to add a trace of an appropriate positive or negative trivalent ion, which will produce immediate clearing. In traces, certain metals such as lead and copper pass into what seem to be colloidal hydroxides by mere contact with water, conferring certain toxic properties on the water. This is known as " oligodynamic " action, of which more will be said later.

Metallic hydrosols can be frequently prepared by reduction of their salts with various reagents, such as phosphorus or formaldehyde in the case of gold. When a metallic salt is hydrolytically dissociated in water, prolonged dialysis removes the free acid, leaving the colloidal hydroxide. Instances are ferric and thorium hydroxides. In such cases, as also in those where the colloid is formed by double decomposition, an adsorption compound with the salt or precipitant is usually formed. For example, ferric chloride on dialysis gives a series of colloids containing, less and less chlorine in relation to iron, from 3 of Cl to 1 of iron, to 1 of Cl to 400 or 500 of Fe, in no stoichiometrical proportion. If dialysis be continued until nearly all the chlorine is removed, the colloid tends to deposit rapidly ; it seems to be stable only when in adsorption combination with a certain amount of the chloride.

To prepare emulsoids free from salts, as is frequently required, the only wav i* prolonged dialysis. Owing to the peculiarity of adsorption being relatively greater the lower the concentration of the solution of the adsorbed substance, it is a matter of much difficulty to remove the last traces of salts (Bayliss, 1906, p. 181). In the case of colloidal dyes, the method of Harrison (1911, p. 17 of reprint) is useful. Precipitate by saturation with ammonium carbonate. This will replace T)ther adsorbed salts. The solution of the dye should be fairly strong, but not too strong, since it will be difficult to filter off the deposit. Probably the use of the centrifuge would be advantageous. Re-dissolve the deposited dye and reprecipitate with ammonium carbonate. Filter again. Repeat the process, if great purity is required. Finally, dry at 110°, which drives off the ammonium carbonate, leaving pure dye. It is important to remember that commercial dyes often contain as much as 30 per cent, of sodium chloride or sulphate.

Matter in the colloidal state is in the form of ultra microscopic particles of solid, or droplets of fluid, in suspension or in other manner of dispersion, in another solid, liquid, or gas. It consists, therefore, in a heterogeneous system, in the sense that there are boundary surfaces of contact between the phases, although these phases cannot be readily separated by mechanical means. Most of the characteristic properties of this state depend on the enormous development of surface in proportion to the total mass.

The chief factor in the stability of such systems, except those of two solid phases, is the Brownian movement of the particles ; this movement is essentially identical with the molecular movement of the medium in which the particles are suspended. There is reason to believe that, by appropriate means, any substance could be obtained in the colloidal state, and that substances usually met with in the colloidal state might be made crystalline. The two great classes of colloids, emulsoid or lyophile, and suspensoid, or lyophobe, which are of the most importance in physiology, differ in the state of the internal phase, which is liquid in the former, solid in the latter. Other properties go along with these, and the names lyophile and lyophobe call attention to the relation of the dispersed phase to the liquid surrounding it. The internal phase in emulsoids frequently consists of a solid substance associated with varying amounts of the solvent, a fact which confers on it the properties of a liquid to a greater or less degree. The relative proportion of water, etc., in the two phases can be changed reversibly by various agencies, especially electrolytes.

The existence of finite particles in many cases can be demonstrated by the ultra-microscope, in which diffraction discs of light, sent off by the illuminated surfaces of the particles, are observed. Owing to the dimensions of these particles, they are unable to pass through a membrane of colloidal substance, such as parchment paper or collodion ; whereas crystalloids pass rapidly through these. The process is known as dialysis and is of frequent use to separate colloids from crystalloids. By the application of pressure, which must be greater than the osmotic pressure of the solution concerned, water also can be forced through, so that this process, known as " ultra-filtration," can be used to concentrate colloidal solutions.

When the internal phase consists of a substance capable of electrolytic dissociation in water, one ion being freely soluble and diffusible, it is found that the surface of the particles is dissociated in this way ; the soluble ions move off as far as electrostatic forces permit, leaving the opposite ions concentrated on the surface of the particle, and giving it their combined electric charges. The giant multivalent ion so formed is called by Hardy a colloidal ion.

The possibility of a source of electrification akin to frictional electricity cannot as yet be definitely excluded as another source of the electric charge, usually found on the contact surface between phases. The possession of this electric charge renders colloidal particles sensitive to the presence of ions of opposite charge. These neutralise the charges on the particles and cause precipitation, themselves being carried down with the precipitate. In this process, the effect of valency is out of all proportion to the increased number of charges. t

In the case of emulsoids, which are less sensitive than suspensoids to this purely electrical effect, neutral salts have a further action, shown in its most marked form as "salting out"; but in lower concentration than necessary for this purpose, they have an action due to their effect on the general properties of water, altering its distribution in the two phases of the system, and therewith other properties, such as surface tension, viscosity, compressibility, coagulation time, etc. This phenomenon may be brought into relation with the association of part of the water with the ions of the electrolytes.

There is also evidence of adsorption of salts in the case of proteins; but whether any true chemical combination occurs is doubtful. Certain emulsoids, such as gelatine, have the property of forming semi-solid structures known as geh. This has been shown in some cases to depend on a redistribution of phases, so that the more solid one changes from the position of internal or dispersed phase to that of external or continuous phase. Another important character of emulsoids is that of imbibition, by which they take up large amounts of water, swelling in the process, and exercising considerable force. Acids and alkalies increase the amount of water taken up in the process. The effect of neutral salts in the main follows the same law as the precipitating action, but it seems necessary to assume an additional factor, probably adsorption.

In imbibition, there are probably two processes at work, one the condensation of water on the surfaces of the colloidal elements, the other, solution of the water in the substance of the particles. No doubt the relative part played by each varies with the amount of water at the disposal of the colloid. Imbibition is incapable of explaining the changes of volume of living cells under the action of crystalloids. Proteins are emulsoids and obey the same laws as other members of the class. As amphoteric substances, they form salts with strong acids or bases, which salts are electrolytically dissociated. In the first case the protein ion, colloidal, is the positive one, so that the particles forming the internal phase will possess positive charges ; in the second case, it will be the negative one.

Since the acidic and basic groups may not be of exactly equal strength, proteins are sometimes naturally electrically charged by surface ionisation of the kind described above. The effects of acid and alkali on the physical properties may be accounted for by the properties of the protein ion, formed in various relative amounts in different cases. Certain proteins are capable of a change, known as "denatu ration," in which their properties approximate to those of a suspensoid, especially in regard to their sensitiveness to electrolytes, in accordance with Hardy's rule of valency.

The phenomena of aggregation and mutual action, presented by mixtures of colloids and crystalloids, offer great complexity and are of much importance in physiological problems, although as yet very inadequately worked out. AN amoeba, after having taken in a vegetable cell, proceeds to digest the substances contained therein. The products, in order to serve as food, must diffuse from the digestive vacuole into the other parts of the protoplasm. But, if they were able to diffuse out from this protoplasm into the water around, they would be lost to the organism. There is good reason to believe, therefore, that there must be some layer or film on the outer surface of an amoeba through which dissolved non-colloidal substances, such as sugar and amino-acids, cannot pass.

Evidence was given in our first chapter to show that living protoplasm must have the properties of a liquid. This fact also points to the necessity of some kind of an envelope, otherwise the organism would stand great risk of colloidal dispersion through the water. The nature of this limiting membrane, with respect to the substances which it allows to pass through, and those which are kept back, is of much importance. It is obvious that a membrane, being merely a thin sheet or film, may be composed of almost any substance. But, for our purpose, it is useful to classify membranes according to their behaviour towards water, and towards substances dissolved in it. In the first place, there are such things as glass or mica, which allow neither water nor substances dissolved in it to pass through. Such may be called impermeable and have a comparatively small importance. There are also some materials which are impermeable to water, but allow certain other liquids or gases to pass through; for example, india-rubber is impermeable > to water, but allows pyridine to pass through. A metal, palladium, may be regarded as impermeable to water under ordinary circumstances, but allows hydrogen to pass through. Such cases are of interest in certain problems.

The most important membranes for the physiologist are those which allow water! to pass through, but hold back dissolved substances. There are various degrees in this respect ; some membranes, such as parchment paper, gelatine, etc., will not allow colloids to pass, but are freely permeable for crystalloids. Copper ferrocyanide, on the other hand, holds back the majority of both colloids and crystalloids, but allows water to pass. A membrane which does not permit any ? dissolved substance to pass, while permeable to water, is known as semi-permeable^ Such a membrane has not been prepared in the laboratory, although the copper! ferrocyanide of Traube approximates to it very closely. When we wish to speak of a membrane which allows water to pass, but not a particular given substance, we say that it is semi-permeable as regards that substance.

Membranes may also be looked at from another point of view, that of their structure. This may be of the nature of a sieve, so that different membranes have different sizes of holes. Or a membrane may allow certain substances to pass through it because of their solubility in the substance of which the membrane is composed. Or, thirdly, they may possibly form reversible chemical compounds with the substance to which they are permeable. The two last cases need not delay us long at this stage. As a case of a membrane which is permeable by a substance, because of the solubility of this substance in the membrane, we may take

a membrane of water, supported in some way, as in wet parchment paper. This allows carbon dioxide to pass through, but keeps back oxygen and nitrogen. Consideration will show, however, that, since these latter gases are not absolutely insoluble in water, after a sufficiently long time there will be no difference in composition between the gaseous mixture on the two sides of the membrane. A membrane of palladium, as investigated by Ramsay (1894), is permeable to hydrogen, but not to oxygen, either because the hydrogen is soluble in it, or because a reversible compound of some kind is formed, which dissociates under a lower

As regards membranes like parchment paper, gelatine, collodion, etc., which allow water t'ind crystalloids to pass, but hold back colloids, it is practically certain that they have a porous structure. Many facts point to this. Biltz (1910) showed that the rate of passage of dyes through parchment paper is in direct relation to their molecular dimensions. Heymans (1912) found that certain micro organisms were able to pass through this paper. When speaking of the ultra-filter of Bechhold, I stated that the permeability could be varied by taking different strengths of collodion, and Bechhold himself (1908) has determined the dimensions of the pores of various membranes by pressing air through them, when covered with water. Schoep also (1911) has been able to control the dimensions of the pores by mixing castor oil and glycerol with the collodion used to prepare the membranes.

The copper ferrocyanide membrane has played a great part in the investigation of osmotic pressure ; the discoverer of it will be of sufficient interest to the reader to warrant the introduction of his portrait (Fig. 44). When a solution of potassium ferrocyanide comes into contact with one of copper sulphate, a membrane in the form of a colloidal gel of copper ferrocyanide is formed at the surface of contact. This gel contains a considerable percentage of water ; if allowed to dry, it becomes impermeable altogether, even to water. In order to be able to perform experiments with such a membrane, it must be supported by being formed in the pores of a cylinder of unglazed porcelain, or, in some cases, in collodion. Further details will be found in the chapter on osmotic pressure. This membrane, although colloidal, obviously has interspaces I >rt \vft-u its constituent elements of much smaller dimensions than those of gelatine or collodion, since, as Traube showed (1867), it does not allow i-anc-sugar to pass through, nor even many salts. Its discoverer regarded it as a " molecular sieve," in that its pores, while large enough to allow water to pass through, were too small to admit dissolved substances. Closer investigation, however, showed that there are some salts which can pass through. Thus, potassium chloride was found to do so, while barium chloride, calcium chloride, potassium sulphate, barium nitrate, and ammonium sulphate could not.

It is pointed out by Ostwald (1890) that it is not necessary to assume that a membrane is impermeable to both ions of a salt, when it is found that the salt in question is not allowed to pass. If one ion only is allowed passage electrostatic attraction on the part of the oppositely charged ions will prevent the permeable ion from travelling further than such a distance at which its osmotic pressure balances the electrostatic force. Copper ferrocyanide is permeable to both ions of potassium chloride; therefore, when it is found to be impermeable to calcium chloride, it must be the calcium ion which is held back. Similarly, in the case of potassium sulphate, it must be the SO4" ion to which the membrane is impermeable.

The fact that the membrane is not completely semi-permeable has led some observers to hold that its permeability or otherwise is a matter of solubility in the substance of the membrane itself. This view does not really lead us any further, and, if we introduce the modern conception of the hydration of solutes, and especially of their ions, it is still possible to look upon the membrane as a sieve. Substances when dissolved become associated with a number of molecules of the solvent, varying with the chemical nature of the solute. Thus, according to J. C. Philip (1907), each molecule of potassium chloride has 7 to 11 molecules of water associated with it, while copper chloride has about 21, and so on. Another fact, which tends to support Traube's view, is that, as he found, a copper ferrocyanide membrane, permeable to potassium chloride, becomes impermeable to it when infiltrated with silver chloride (Traube, 1899, p. 261). It does not seem likely that there should be any material difference between the solubility of potassium chloride in silver chloride or in copper ferrocyanide ; if any, one would expect it to be more soluble in the chloride, according to the old law, " similia similibus solvuntur" (Rothmund, 1907, p. 112). On the other hand, it is to be presumed that any pores present would be narrowed by deposition of silver chloride on their walls.

A detailed investigation of the permeability of a large number of precipitation membranes was undertaken by Paul Walden (1892). If the table on pp. 716 and 717 of his paper be consulted, various facts will be noted which have a bearing on the question before us. The membranes can be arranged in order of merit, as regards impermeability to the substances tested. Tannin-gelatine is the lowest in the series, being permeable to all -except tannin itself ; while copper ferrocyanide is the highest, being impermeable to a larger number than any of the others. A significant fact is that none of the membranes comes out of its place as regards any particular substance. That is, assuming that the pores increase regularly in dimensions from the copper ferrocyanide to the tannin-gelatine, no substance is found which diffuses through a membrane having the smaller pores while being held back by that with the larger pores, as might happen on the solution theory. The behaviour of the hydrochlorides of the three ethylamines is of interest. The copper ferrocyanide membrane is readily permeable to that of monoethylamine, slightly permeable to that of the diethylamine, impermeable to that of the triethylamine, following the increase of molecular dimensions.

The difficulty frequently arises, however, as to the proof that the membrane is not chemically acted upon, or injured in its integrity, when it appears to be permeable to a particular solute. This consideration seems to deprive Tammann's experiments with dyes (1892, p. 257) of much of their value, although this observer draws the conclusion that there are dyes which pass a membrane which is supposed to have the smaller pores, while bein,g held back by one with the larger pores, and that Traube's theory does not hold. In Walden's experiments, the permeability of the membranes composed of the ferrocyanides of zinc and of copper is identical, whereas in those of Tammann the zinc membrane shows itself to be permeable to dyes to which the copper one is not ; it is even stated to be permeable to " Baumwollenblau " to which the tannin-gelatine membrane is impermeable, and even parchment paper only slightly so. If we neglect quantitative differences, which are very difficult to judge satisfactorily, there are only two out of Tammann's seventeen dyes which fall out of line. " Baumwollenblau " is one of these and the other is fuchsin-chloride, to which copper ferrocyanide is permeable, zinc ferrocyanide not. According to Cain and Thorpe (" Synthetic Dye-stuffs," 1913) "cotton-blue" is a mixture of ammonium and sodium salts of di- and trisulphonic acids of rosaniline blue. Since even parchment paper is impermeable to the salt of the mono-sulphonic acid ("aniline-blue ") it is difficult to believe that a zinc ferrocyanide membrane (if perfect) should be permeable to the " cotton -blue " mixture. The experiments of Biltz (1910, p. 117) on the passage of dyes through parchment paper, have been referred to above. These experiments show an unmistakable relation between the molecular dimensions of the dye and its ability to pass through the paper.

If the number of atoms is less than 45, it passes through quickly ; above 45, slowing begins to show itself ; between 55 and 70, the passage is very slow ; and about 70, it ceases altogether. Of course, the actual space occupied bv a molecule does not depend only on the number of atoms it contains. The chemical arrani;i'iin nt must al>"> l>e taken into account ; accordingly, chemical structure was found to have some effect on the results. The "sieve theory," then, appears to hold in the case i.f < "lloids and, as we cannot draw a lino of demarcation between them and iT\>tallnids, the general application of the theory receives support.

Abel ( 1914) finds in his " vividiffusion " method, that the rate of diffusion through collodion membranes is independent of their thickness, a fact which suggests pores rather than solution in the substance of the membrane. When we recollect that the copper ferrocyanide membrane is freely permeable to water, in fact, contains water in its constitution, it seems not so easy to understand how a substance such as sugar, which is easily soluble in the water

-contained in the membrane, fails to pass through, unless something like a sieve is present, opposing a mechanical constraint on molecules above a certain size. Traube (p. 280 of the "Collected Papers") points out that his precipitation membranes always contain considerable quantities of water, and that, if dried, they become completely impermeable, both to water and to solutes. Bartell (1911) showed that, when water is forced by pressure through a membrane of copper ferrocyanide, the rate at which it flowed through obeyed Poiseuille's formula for the case of capillary tubes.

But, before the question at issue can be finally decided, it will be necessary to understand more completely the nature of the process of solution, and it may very probably be found that there is no real contradiction between the two opposing views. With regard to the structure of colloid membranes in general, it will be clear that the remarks on page 14 above are of importance. If a membrane of gelatine has a honeycomb structure, any substance passing through it must traverse a structure consisting of much finer pores than if the membrane were of a sponge-like nature, where it could pass, by a tortuous channel, between the actual trabeculse of the solid phase.

Another point to be remembered is that the surfaces of the elements of the / membrane adsorb dissolved substances. In the filtration of salts through a gelatine filter, the first portions of the filtrate contain less salt than the original solution ; this continues until the adsorption capacity of the membrane is saturated. A colloid, when adsorbed, may diminish considerably the dimensions of the pores, so that the filter becomes impermeable for substances to which it was at first permeable.

It is frequently found that a solute, to which a membrane appears to be impermeable, will pass through in very small amount, if allowed a long time. Theie are two possible causes for this fact. The pores in an artificial membrane are not all of exactly the same size, as was noticed by Bechhold in his measurements of various membranes. Suppose that there are a few of them which will allow a certain solute to pass, while the great majority are impermeable to it ; it will take a long time for an appreciable amount of the solute to find the small number of channels available for it, owing to the slowness of diffusion. A similar state of affairs would be found if the particles of the solute varied in dimensions, even if the membrane were of a uniform structure.

These facts lead to reference to the rate of passage through ;i membrane. In addition to the factors mentioned in the previous paragraph, a little consideration will show that a membrane may be freely permeable to a solute, but, if the rate of diffusion is very slow, comparatively little will pass in unit time, owing to the supply at the surface of the membrane not being kept up sufficiently. This state of affairs plays a part in certain osmotic phenomena to be discussed in the next chapter.