Principles of General Physiology

We cannot, of course, actually perform the operation in the manner described. In an indirect way, however, it was done by Faraday (1858, p. 159), who found that, by acting on solutions of gold salts by reducing agents, beautiful red or purple solutions were obtained. He also showed that these solutions, although permanent, were, in reality, suspensions of minute particles of metallic gold (p. 160 of the above paper). It is interesting to note that one of Faraday's gold preparations is still preserved in the Royal Institution.

Since these gold solutions have served as the foundation for much subsequent work, the method of preparing them is worth description. The ruby-red solution is made thus, in the words of Faraday himself (1858, p. 159) : " If a pint or two of the weak solution of gold before described" (i.e., about 2 grains of gold chloride in two or three pints of water) "be put into a very clean glass bottle, a drop of the solution of phosphorus in sulphide of carbon added, and the whole well shaken together, it immediately changes in appearance, becomes red, and being left for six to twelve hours, forms the ruby fluid required ; too much sulphide and phosphorus should not be added, for the reduced gold then tends to clot about the portions which sink to the bottom." Zsigmondy (1905, pp. 97-101) finds that the method is improved by the addition of potassium carbonate, in order to neutralise the free acid produced in the reaction ; he also gives other useful hints, pointing out the importance of pure water and Jena glass vessels ; the absence of colloidal matter from the water used appears to be especially necessary if uniform results are to be obtained. The necessity of cleanliness was well known to Faraday himself, although at that time the properties of colloids were unknown.

A beautiful deep blue solution of gold can be made by reduction with hydrazine hydrate (Gutbier, quoted by Svedberg, 1909, i. p. 10). Gold chloride O'l per cent, is neutralised by sodium carbonate and very dilute hydrazine hydrate (one part in 4,000 of water) added drop by drop, carefully avoiding excess. How do we know that we have to do with suspended solid particles in these preparations ? They are quite transparent to light of ordinary intensity, although this does not apply to all colloidal solutions ; where the particles are larger the solutions are turbid, and their appearance suggests their nature. Even the most transparent gold preparations, however, were found by Faraday to show turbidity in the track of a powerful beam of light. This observation forms the foundation of the ultra-microscope, to be described later. It is frequently called the " Tyndallphenomenon," but its discovery was really made by Faraday (1858, p. 160). Tyndall pointed out that the light reflected, or rather diffracted, from the path

of the beam is polarised, a fact which proves that the particles are of the same order of dimensions as the mean wave length of the light used. A further proof that we have to do with suspended particles is given by Friedenthal (1913). By powerful centrifugal force, he has separated several colloids from solution, caseinogen from milk, for example. Iodised starch, mixed with non-iodised, could be separated from the latter, owing to its greater weight.

The colloidal state, then, is of the nature of a heterogeneous system, or a system of more than one separate phase. The point of importance to be remembered is that the phases of which the system consists are separated from one another by surfaces, interfaces, of contact. The colloidal state differs from a coarsely heterogeneous system, such as a mass of gold immersed in water, in that it is, to ordinary observation, homogeneous, and only shows its micro-heterogeneous character by special methods of investigation. On the other hand, it is distinguished from true solutions of small molecules or ions by the fact of the possession of surfaces of contact, with all the phenomena implied by this. These properties will naturally be especially marked on account of the great surface area due to the minute state of subdivision.

It is convenient to have names for the two phases of which a colloidal system usually consists. If we refer back to Fig. 15 (page 14), we see the appropriateness of Hardy's names (1900, 2, p. 256), of "external" and " internal " 'phases. Other workers call Hardy's internal phase the " dispersed phase," and the external phase the "continuous" one (Wo. Ostwald, 1907, p. 256). The names will be used here indifferently. One essential condition for the production of a colloidal solution of a substance is that it should be practically insoluble in the external phase, or " dispersing medium," to use another expression of frequent usage. This statement, however, needs some qualification, as we shall see later. It is especially insisted on by von Weimarn (1911, p. 6) that, given appropriate conditions, all substances can be brought into the colloidal state.

It may be mentioned, as an illustration, that resinous substances like gamboge or mastic form true solutions in alcohol, but when such solutions are poured into water, a colloidal solution is produced. The same investigator gives strong evidence to show that, conversely, all substances can, by appropriate manipulation, especially very slow deposition, be obtained in the crystalline form (1912) ; although the crystals of such liquid or semi-liquid substances as proteins are apt to be very minute and distorted in shape, rounded at the edges, by the action of surface tension.

Most of our knowledge of the fundamental properties of the colloidal state is due to Thomas Graham, whose portrait will be seen in Fig. 35. Graham started from a different point of view from that of Faraday. He noticed that certain substances are extremely slow to diffuse, and devoid of the power to crystallise (1861, p. 183). They are also unable to pass through a membrane of similar nature to themselves, such as sized paper or parchment paper (unsized paper treated with sulphuric acid). Amongst these substances are hydrated silicic acid, starch, albumin, gelatine, etc. He says (1861, p. 183): "As gelatine (KoAX?/ = glue) appears to be its type, it is proposed to designate substances of the class as colloids, and to speak of their peculiar form of aggregation as the colloidal condition of matter. Opposed to the colloidal is the crystalline condition. Substances affecting the latter form will be classed as crystalloids The distinction is no doubt one of intimate molecular constitution." It will be noted that, although Graham speaks here of the " colloidal condition " of matter, he appears to regard the class of colloids as quite distinct from that of crystalloids. "They appear like different worlds of matter" (1861, p. 220). At the same time he is aware that the same substance, silica for example, may be obtained in either state, while on the page following that on which the above statement is found, he suggests that the colloid molecule may be "constituted by the grouping together of a number of smaller crystalloid molecules." Perhaps stress is intended to be laid rather on the word "appear." In any case, it is better to speak of the " colloidal state " and not of " colloids " as a class.

One important characteristic of this state, that of instability, was clearly recognised by Graham. After referring to the fact that colloidal solutions of The signature is taken from the facsimile reproduction of the Charter Book of the Royal Society. silica sooner or later become gelatinous and finally crystallise, he says (1861, p. 184) : "The colloidal is, in fact, a dynamical state of matter; the crystalloidal being the statical condition. The colloid possesses ENERGIA. It may be looked upon as the probable primary source of the force appearing in the phenomena of vitality. To the gradual manner in which colloidal changes take place (for they always demand time as an element), may the characteristic protraction of chemico-organic changes also be referred." This "energia" we know now as "surface energy" of its various kinds.

The two phases of which a colloidal solution consists may obviously be of many various kinds. The table below will illustrate this: — The most important systems for the physiologist are those consisting of solids and liquids, Nos. 3, 4, and 6. The nature of the dispersed phase as solid or liquid has been adopted as a basis of classification by Wo. Ostwald (1907, p. 334). This system is in many ways a useful one, although it does riot direct attention to what is perhaps the most important distinction between different classes, that is, the affinity of the dispersed phase for water. When the internal phase, although liquid, is in extremely minute droplets, its mechanical properties closely resemble those of a solid — the great pressure due to the internal component of the surface tension confers rigidity on them. The characteristic which carries with it most of the other differences in the general behaviour of a colloidal system is the affinity of the internal phase for water, or other solvent, constituting the external phase. It will be clear that the more water the internal phase contains, and it may contain as much as 90 per cent., the less will be the difference between the properties of the two components of the interface of contact between it and the external phase, and, consequently, the less will be the surface energy.

Hardy (1900, 1, p. 236) calls attention to the fact, also pointed out by Quincke (1902, p. 1012), that, as a rule, the material of which the internal phase is composed is not absolutely insoluble in the external phase, so that the two phases will be (1) a solid containing a certain amount of the solvent, and (2) a very dilute true solution of the solid. The substance of whicli the solid phase is composed will become more soluble, as a rule, as the temperature is raised. This fact is sometimes of use as a means of indicating whether the external phase of a colloidal solution does actually consist of a dilute true solution of the substance in suspension. The most convenient way of detecting this is by measuring the electrical conductivity. However long a colloidal solutionhas been dialysed (a means of purification to be described later, and depending on the impermeability of certain membranes for colloids), it is almost impossible to remove all traces of foreign electrolytes. Now, as the temperature is raised, these foreign bodies will not increase in number ; since the impurity is in extremely low concentration, it may be regarded as being completely dissociated electrolytically. The increase in conductivity, so far as it depends on this impurity, will be due only to the increased rate of movement of the ions already present, dependent on the diminished viscosity of the solvent. The temperature coefficient of this is known, and lies between 2 and 2-4 per cent, of the conductivity at 18° per degree rise of temperature. Suppose we take a solution, saturated at 18°, of an electrolyte, for convenience a somewhat insoluble one, such as sulphanilic acid, and determine its conductivity at various temperatures, we find the temperature coefficient to be 2'6.

But if excess of undissolved acid is present, more and more will go into solution as the temperature is raised, the actual number of ions is increased, and the temperature coefficient appears to be considerably higher, viz., 5 '9. Applying this fact to the colloidal system, if the conductivity is due to foreign ions, the temperature coefficient will be only 2 to 2'4, and if it is found experimentally to be higher than this, evidence is afforded that more of the colloidal substance itself goes into true solution. The free acid of Congo-red is a

case in point. Urn-, as I rind, tlie temperature coefficient is either :>•<; <\r 7'.'», according to whether the measurements are made from higher to lower, or vice versa. The difference is, no doubt, due to hysteresis (see below). The measurements were made in a quartz vessel. The hypothesis can be tested in another way, not so satisfactory in practice. If a dilute solution of an electrolyte be further diluted, say to twice its volume, its conductivity will be halved, because no new ions will be produced. If the conductivity of a colloidal solution be due to traces of electrolyte impurity, on dilution its conductivity will be reduced in exactly the same proportion. Whereas, if due to slight true solubility of the colloid itself, it will remain unaltered ; or at all events, less diminished than in ratio to the dilution. Tln-r«- is always excess of the solid phase present, so that the external phase is always a saturated solution. If the particles diminished in size, owing to further subdivision, greater dispersion,

milli moles per litre. This fact is in agreement with the experience of chemists that large particles in precipitates grow at the expense of smaller ones ; or from a mixture of crystals, deposited from a hot saturated solution when it cools, the smaller crystals gradually disappear while the larger ones increase in size. The fact is connected with the diminution of surface energy involved in the process. Perrin (1905, p. 85) divides colloidal solutions into "hydrophile" and "hydrophobe," according to the affinity of the dispersed phase for the water ; " lyophile " and "lyophobe" would be better, as Freundlich points out, since water may be replaced by other solvents. This classification is almost coterminous with that of Hardy (1600, 1 and 2) into reversible and irreversible colloids, according to whether, after evaporation to dryness, they go intq solution again on mere addition of water or remain as a solid film. Typical instances of the hydrophile class are gelatine and gum, of the hydrophobe class, gold and arsenious sulphide. Intermediate forms are also known to exist, that is, systems which have some of the properties of each class. Such are the sulphur preparations of Sven Ode'n (1912, p. 712), which give reversible precipitates with salts, like the hydrophile class, but are precipitated by very small concentrations of bivalent ions, like the hydrophobe class. It must be admitted that the existence of these intermediate kinds of colloidal systems deprives all classifications as yet proposed of much of their theoretic value, although useful in practice.

The names "sol" and "gel" introduced by Graham (1864, p. 321, p. 620 of the Collected Edition, 1876) may be referred to here; a colloidal solution of silicic acid, at first liquid, becomes gelatinous in process of time. The two states are called "hydrosol" and "hydrogel" respectively, when the external phase is water. When this is alcohol, " alcosol," and so forth. Some degree of confusion is apt to arise from the use of the words "homogeneous ' and "heterogeneous" as applied to solutions. It is plain that no solution can be absolutely homogeneous ; a molecule of water and one of sodium chloride cannot be in the same place at the same time. Indeed, von Calcar and Lobry de Bruyn (1904, p. 218) thought that they had succeeded in producing, by centrifugal force, changes of concentration in solutions of potassium iodide. It is also clear that, if we make as our criterion of heterogeneity the power we possess of separating the phases mechanically, as Bakhuis Roozeboom (1901, p. 9) does, colloidal solutions cannot be called heterogeneous. The really important point is, following the work of Willard Gibbs, whether the phenomena due to the possession of surfaces of . contact, as shown by matter in mass, are also shown by the "particles" of the iiiternal phase in colloidal solutions. About this there is no dispute ; but, to avoid misunderstanding, it is perhaps advisable not to use the name " heterogeneous " in their case, and to speak of colloidal solutions as " micro-heterogeneous," one or more of the phases being minutely subdivided.

Where then can we say that " molar " properties cease and " molecular " properties begin ? The question remains as yet unanswered, but it seems clear that a gradual transition must exist, and possibly some of the disputes as to the relation between the chemical and physical properties of certain colloidal systems may lie due to an exclusive consideration of a part only of the phenomena shown by these intermediate states. Whatever phenomena are manifest at interfaces between phases will obviously be greater as these interfaces increase in area. It is of interest, therefore, to calculate the amount by which the surface of a given mass increases when subdivided to colloidal dimensions. The particles of gold in some of the preparations of Siedentopf and Zsigmondy (1906) were found, by a method to be described

later, to have a radius of about one-millionth of a centimetre. A sphere of gold of onetenth of a centimetre radius has a surface of 0-126 sq. cm., while the surface of the same mass, if subdivided to the above colloidal dimensions, would have a surface of about 100 sq. m., or be multiplied by ten millions. It will occur to the reader that we are very near molecular dimensions in the case of these finest particles. In fact, Siedentopf and Zsigmondy obtained gold hydrosols with particles of less than 6 ^ in diameter (fj. is O'OOl mm., and /JL/J. is one-thousandth of this, i.e., onemillionth of a millimetre), while starch is stated by Lobry de Bruyn and Wolff (1904) to have a molecular diameter of 5 iJ.fi, and even carbon dioxide has a value of 0"29 fn/j. (Nernst, 1911, p. 434).

In practice it is found that Graham's criterion of not passing through parchment paper is the most satisfactory one for deciding whether a particular solution is a colloidal one. This property goes together with the various other properties dependent on surface development, although it must be admitted that it is somewhat arbitrary to fix the point at a definite dimension. Indeed, there are substances on the border line, like certain dyes, which will pass through some samples of parchment paper, but not through others, and these substances are found to possess some of the colloidal characteristics but not all.

When we are dealing with such things as gold, silica or arsenious sulphide, we know that the size of their particles can only be attained by the aggregation of a number of molecules ; but, as we have just seen, the single molecules of some organic compounds, such as starch, may be of sufficient size to present properties of surface. Haemoglobin does not pass through parchment paper, but measurements of its osmotic pressure by Hiiffner and Gansser (1907) have shown that it is present in solution in single molecules. How this is known will be understood after Chapter VI. on osmotic pressure has been read. In the case of salts, such as Congo-red or caseinogen in alkaline solution, which are electrolytically dissociated in solution, but of which neither ion passes through parchment paper, complications are present which will be discussed in the next chapter. It may be that the organic ion itself is sufficiently large to possess the properties of the colloidal state, or there may be aggregates of these ions formed.

Much of the recent progress in knowledge of the colloidal state is due to the use of the ultra-microscope. This method was first described by Siedentopf and Zsigmondy in 1903. Details of the construction of the instrument would be out of place here. The reader is referred to the original paper (1903) or to the book of Zsigmondy (1905, pp. 83-97). Space for the principles only, on which it depends, can be found here. It is a matter of common observation that dust particles, completely invisible under ordinary light, become clearly visible in a beam of sunlight. Rayleigh (1899) has shown that to make visible a particle, which is too small to be seen by the highest power of the microscope, merely requires sufficiently intense illumination. It must be remembered that these particles are smaller than the wave lengths of the visible part of the spectrum. For example, the wave length of the D line of sodium is 589 p.^ and the limits of the visible spectrum lie roughly between 700 and 400 pp. Dimensions of such values are high for the particles in a colloidal solution, which may be as small as 6 /xju., as we have seen, although this is an unusually small size. Any object smaller than half the wave length of the light by which it is illuminated cannot be seen in its true forjn and size owing to diffraction. Hereby is set a limit to microscopic observation. A brilliantly-illuminated dust particle in a beam of sunlight is seen as a disc, due to diffracted rays sent off from its surface, and looks much larger than it actually is.

The Faraday phenomenon in a colloidal solution is similar to that of the motes in a sunbeam. It occurred to Siedentopf and Zsigmondy that if the solution was much diluted and the beam examined by the microscope, placed perpendicularly to its track, so as not to receive the direct light, the diffraction images of the separate particles would be visible. In that form of the ultramicroscope made for the examination of liquids, a very intense but small beam of light is projected horizontally from the sun or an arc lamp, by means of a system of condensing lenses, into the liquid contained in a small cell with a flat side towards the light and a flat top towards the observer. The track of the beam is examined from above by means of a water-immersion lens forming the objective of an ordinary microscope. If the solution contains particles, these are seen as bright discs with vigorous Brownian movement. The limit of visibility depends on the intensity of the illumination. The finest particles cannot be distinguished separately, but are indicated by a haze. Zsigmondy uses the name, " submicron," for elements seen as separate discs, although invisible in the ordinary microscope, and " amicron " for those which even the ultra-microscope can only indicate as a diffuse illumination in the track of the beam. Fig. 36 shows the course of the light rays ; Fig. 37 the arrangement of the apparatus, in Zsigmondy and Bachmann's (1914) pattern, and Fig. 38 shows the illuminated field seen by the observer.

The correct interpretation of all the phenomena seen by this method has not been arrived at as yet, and much caution must be exercised in drawing conclusions. There are one or two points which a little experience in its use with a variety of solutions has taught me, that it may be well to call attention to. It is a matter of some difficulty to obtain water that does not show a few particles, so that in the preparation of colloidal solutions foreign particles are almost unavoidable. Now these may be mistaken for the substance under examination. To take an example, a dilute solution of Congo-red, even the purest, is almost certain to show a few bright discs of light ; but, on close observation with the most intense light that the apparatus can give, it will be noticed that the track is filled by a faint haze. In this case it can easily be shown that the few particles seen are not the dye itself ; the addition of a little acid to the solution splits off the free dye acid and this forms a colloidal solution witli comparatively large particles, so that the whole track of the beam appears densely packed with bright diffraction images. It is unnecessary to repeat that this appearance of denselypacked particles is really due to the disparity in size between the objects and their diffraction images. What is clear is that the dye salt is not resolvable into particles, it consists of "amicrons," whereas the free acid consists of " submicrons."

If a given solution cannot be resolved, it must not, therefore, be assumed that it is a true solution. It may consist of particles too small to be seen by the available illumination, or their refractive index may be too close to that of the surrounding medium. In the actual instrument, as supplied by the firm of Zeiss or Winkel, for example, the crater of the positive carbon of the arc is first focussed on to an adjustable slit, whose aperture can be read off on the graduated head of the adjusting screw. This slit can be rotated through 90° for the purpose of estimation of the actual mass of the particles by the ingenious method of Siedentopf and Zsigmondy (Zsigmondy, 1905, pp. 93-97). The total content of the colloidal matter in a lar-v volume of the solution is first determined by some appropriate chemical method. This solution is then diluted to a known extent, and so far that the particles seen under the ultra-microscope are sufficiently separated to be counted. By aid of a

micrometer in the ocular, a known area of the field is isolated, and the number of particles in the volume corresponding to tlii.x area is counted. The depth of this portion is obtained by rotating the slit through 90°, when what was previously the depth becomes the width, and can be read off on the ocular micrometer. A simple calculation then gives the number of particles in unit volume of the original solution, and from this the mass of each is known from the total solid content of the solution.

The rays converge to a focus in the centre and then diverge again, a and b. Note that the greatest number of particles is rendered visible in the most brightly illuminated spot c. This is due to the fact that the more intense the illumination, the smaller are the particles that it is possible to observe. The particles which are too small to be seen outside the focus of the beam are obvious under the more brilliant light at this focus. "ultra-microscopic," which is frequently used for examination of bacteria, and is then, of course, not strictly ultra-microscopic, but can also be made to show the presence of structures invisible by the ordinary microscope, is a development of the dark ground illumination by specially constructed sub stage condenser, introduced by Wenham in 1872. The central rays of the illuminating beam are cut out by means of a stop, and the peripheral rays are reflected by a parabolic surface so as to meet at a point in the object under examination ; they cross at such an angle as to pass outside of the field of the objective in use, which only picks up light refracted, or diffracted, from structures in the preparation. The paraboloid form is chiefly used for the investigation of comparatively coarse structures, as in Fig. 6, of Spirogyra. A cardioid surface, as in the apparatus of Siedentopf, made by Zeiss, gives more brilliant illumination, and can be used for the more minute particles of colloidal solutions. This latter instrument has also been fitted, at my suggestion, with an electrical hejting arrangement, so that the changes produced in colloids by heat can be followed by the eye.

The scattering of light by suspended particles has been made the basis of a method of estimation by Theodore W. Richards (1906 ; also Biltz, 1907). Accurate determinations of small amounts of precipitates can be made in this way. The instrument used is called, by Richards, " Neplidometer." One definition of the colloidal state is that, matter in this state does not pass through such a membrane as parchment paper. The discovery of the fact is due to Graham (1861, p. 186), as well as the application of it to the separation of colloids from crystalloids by the process which he called "dialysis." The forms of apparatus which he used are shown in Fig. 39, and are in practice very effective.

I find that it is better not to allow the level of the liquid inside to rise above the upper edge of the paper, since it is difficult to make a tight joint at the lower edge of the hoop or glass bell. The sheet of paper taken should be large enough to be tied around the top of the vessel. A continuous current of water may be caused to flow through the outer vessel, but a given volume of distilled water is more effective if used in several changes of the whole volume of liquid in the outer vessel.

Crystalloids pass very rapidly through parchment paper. Graham showed that 96 per cent, of the salt content of a 2 per cent, solution of sodium chloride passed through in twenty-four hours, when the volume of the water outside was ten times that of the solution and was changed once. Dilute hydrochloric acid applied to one side of the paper reddened litmus paper on the opposite side in 5 '7 seconds. A point to be remembered is that the paper itself is altered by the action of alkali, expanding more than by the action of water alone. This will affect its permeability, and, in fact, I have noticed that Congo-red, which passes very slowly through some samples of the paper, is accelerated in this process if the solution is slightly alkaline.

Other forms of dialyser will be found described in the practical handbooks, such as the article of Zunz (1912, pp. 478-485). J. J. Abel (1913 and 1914) has applied the process of dialysis to the investigation of chemical changes occurring in the whole organism of the higher animals or to those occurring in individual organs. The blood, issuing from an artery through a canula, is made non-coagulable by the addition of small amounts of extract of the heads of leeches, run into it from a side tube, and is then caused to pass through a series of collodion tubes, immersed in warm Ringer's solution. Collodion, like parchment paper, is impermeable to colloids. After passing these tubes the blood is returned to a vein and thus is kept in continuous circulation through the dialyser. In its passage, it gives up the diffusible substances which it contains to the outer fluid, in so far as they are not already present in equal concentration therein. By sufficiently long continuation of the process, these substances pass out until they are in equal concentration in the blood and in the outer liquid. If the maximum degree of dialysis is required m a

limited time, the Ringer's solution is changed at intervals. Abel has already obtained considerable amounts of amino-acids. To investigate the changes taking place in the contents of the blood as it traverses a particular organ, the diffusate from the ingoing blood can be compared with that of the outgoing blood. The substances that have been identified as diffusing out from the blood are sugar, urea, phosphates, amylase, and amino-acids. The name of vim-diffusion is given to this method by its discoverer.