FURTHER STUDIES I N PERIODIC PRECIPITATIOY BY FRASCIS E . LLOYD AND VLADIMIR X O R A V E K
During our studies leading to the publication of a previous paper (1928) the authors were impressed by the wide variety of behavior and the static forms of precipitate resulting from interaction of reagents in fluids and gels to such an extent that it was determined t o make as extensive a survey as possible of all systems in which periodic precipitation might occur. We believe it necessary before a comprehensive generalization can be formulated to make a broad survey and to acquaint ourselves with all possible behaviors and their results; and as such results cannot be adequately described verbally, we adopted the method of record by careful photography and cinemaphotography. Indeed the complexity of the subject is so great that nothing short of visual experience can make the variations of behavior comprehensible and especially since many authors have not made adequate records of their experiments-few of the photographs published show clearly what they are intended to show and are often very poor and unconvincing-and because most if not all papers hitherto published advance theories based on one or a fern experiments with a single system only. Their authors have frequently lacked perspective to he gained only by a wide survey.
1. The E.fect of Spatial Relations. We have found that many experiments done, as is usually the case with test tubes show no periodicity for hours or days, or even not at all, whereas the same experiments done in capillary tubes show periodicity within the first few minutes of experimentation. This is in many cases due t o the enhanced if also microscopic visibility due to the smallness of the masses of precipit,ate. Severtheless, we have already found other effects due to the small extent of the space in which the reactions proceeded (1928) and we therefore tried to determine more exactly to what such effect might be ascribed. Two such effects have been noticed, viz. ( I ) that on the rate of diffusion and (2) that, resulting from adsorption on the walls, when adsorption occurs. diffusing into i\lgC12-gelatin we found that in a . In the system ”,OH the lowest concentrations no periodicity could be observed in test tubes, while in capillary tubes, well-marked, widely separated, periodic massive bands occurred. (Figs.’ I,, 3). This is due to the slowing up of diffusion rate in the capillary tubes, which measured . j to I mm. in diameter. Under these conditions the OH-ions being negatively adsorbed by the glass wall move forward in the middle of the column and the Mg-ions move radially to meet the OH-ions. The precipitate therefore takes that form of a spindle, more especially the smaller the tube and therefore the greater the curvature. I n All “figs.” refer to figures in the plates. Text figures are so designated
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the inverse system MgC12diffusing into NH,OH-gelatin (Figs. 2 , 4), marked irregularities also occur in both test tubes and capillary tubes, but here because the Mg-ions are positively adsorbed the bands usually fill the lumen, but are of irregular form, the negative adsorbability of OH playing a minor r61e, and, in the test tubes especially, the questionably periodic masses of precipitate are very irregular, due to local impermeability of the initial precipitate plug, and are attached strongly to the glass wall, being adsorbed thereto. In this system also the differences as between test tubes and capillary tubes are very marked; in the former usually no periodicity is observable, while in the latter it is well marked. When we repeated the above experiment, we obtained in one concentration, curiously enough, a very regular distinct band. (Fig za, Plate IX). This does not invalidate the general truth of our conclusions. (Figs. 1-4). b. Also in the system 20% NHIOH into 2 5 CoC12in 10% Difco gelatin in tubes I , 3 , I O and j o mm. in diameter, we found that in the narrower tubes the white precipitate was more extensive and colored bands wider and the whole diffusion column shorter than in the widest tube. In the latter there was little or no white precipitate, while in the capillary tubes the spaces between the bands were filled with it and a broad band of white precipitate was produced in advance of the last blue colored band. The tubes of intermediate diameters showed intergradations of behavior. (Fig. 8). c. Effect of convection currents. In large tubes, because of convection currents and gravitational effects, it is impossible to obtain evidence of periodicity in fluid media. When, however, we enclosed Pb(xO3)2 diffusing into KI (or the inverse) in a very narrow space between a slide and cover glass, well marked bands of crystalline PbI2 were obtained. (Fig. 7 ) . The same result was obtained in .5K Pb(SO3)z into .5X SaCI. (Fig. I I O ) . d. In the system . z 5 P ~ J ( N O diffusing ~)~ into .IN-.oI2j?: K2Cr20i in gelatin in capillary tubes 3 mm. wide it was not possible to observe periodicity except in microscopic sections. In a very thin layer of gelatin formed by the pressure of a large bubble beautiful bands were developed. In this instance the resulting forms afforded a charming spectacle (Fig, 9). e. The trichomes of plants afford minute capillary spaces of tubular form and in such we have obtained clear and visible periodicity with the system S H , H S into Ka3Co(KOz)6, Moreover, the precipitate occurs within the cellulose wall itself, not in the lumen except toward the end of the trichome. This can be due to the washing out of the free reagent, since the preparations were repeatedly treated with cold distilled water before adding KH,HS (Lloyd, 1925) (Figs. 5, 6, IO). In tubes of much greater calibre periodicity occurs or a t least becomes visible only after a relatively long time. (Lloyd and hloravek, 1928). f. To be mentioned here is the example set forth by us in 1928, that in the same system as in (e) above only rings of precipitate are to be obtained in minute capillary tubes, whereas in larger tubes rings and discs or saturn structure are formed.
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FRANCIS E. LLOYD AND VLADIMIR MORAVEK
From the foregoing it will be seen that clean-cut periodicity may be procured in large spaces, but obscurely or not at all in small ones and, vice versa, periodicity may occur in small spaces and not at all in large ones. Such behaviors are indeed secondary in nature demanding special explanation, for it is clear that in many systems the tendency to periodicity is always and indubitably present but may be obscured by the special conditions present. The importance of this from a biological point of view can hardly be overestimated since many interactions must occur in restricted spaces, often of irregular shape. The now familiar case of the “Fromman striations” on the treatment of medullated nerve fibers with silver nitrate “is the result of the operation of physical processes which in capillary spaces produce the Boehm (or Liesegang) phenomenon.” ( A B . Macallum, 1906).
I I . Nature of the Medium. It has often been thought that the nature of the medium determines in a primary sense the incidence of periodicity. This, however, is not true, as many experiments in the literature, as well as our own, prove. As Doyle and Ryan correctly observe, an explanation of the phenomenon which attributes a fundamental r81e to a gel as a medium cannot be correct. We may regard this aspect of the case as a closed issue. We cannot therefore agree with Bradford that “the nature of the gel is of fundamental importance,” but rather take the position that the effect of the gel in each case must be explained and that also the r81e of the gel is a purely secondary matter. Concerning the theory of Dhar (1922) and his associates, we have t o say that it assumes the presence of a gel or other peptizing substance. The secondary influence of the gel medium has been discussed and demonstrated experimentally very successfully by Wo. Ostwald (I 926) contra Chatterji, Dhar and Dogadkin. We think that McGuigan and Brough (1923/4) are mistaken when they declare that “no new chemical process is involved in the formation of rings,” and when further they attach a dominant value to the dampering effect of the medium. Dampering effect there is, but it plays a secondary rble, and i t would be difficult according to their views to explain why a periodical system should occur in water and not in gelatin. See also Rouppert, whose paper contains pertinent citations (1926). Rouppert cites the important fact that periodicity can be obtained with gases only if, water vapor be present, as observed by Chapin and Holmes (1918)~E. Karrer (1921) and Fischer and McLaughlin (1922). We have made use of gaseous, fluid, gelatinous and solid media. The most obvious and indeed the most important practical character of gaseous and fluid media is their instability and the consequent instability of the whole system. On the other hand, a gel supplies a framework which can stabilize the system and thus prevent mass movements which obscure or obliterate the results. This can be regarded as obvious, but it cannot be stated as a corollary that the higher the viscosity of the medium the more certain periodicity is to
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occur. Our experiment shown in Fig. 3 j illustrates this point and will be referred to beyond under concentration of gels. Periodicity depends rather upon the amount of material (which can be expressed as dry weight) present and not a t all on the viscosity of the medium. This conclusion makes it possible to understand more clearly the comparative behavior of a particular reaction system in different gels. We have worked with gelatin, agar and starch. It is well known that agar preserves its inhomogeneity and substantial character in spite of heating a t boiling temperatures, and starch still more so. We may now consider the effect of the various media used, viz. gaseous, fluid, solid and gel. Gas:-R. B. Peet (1925) in Young's laboratory experimented with HCI and XH3. He experienced much difficulty which we avoided by the use of capillary tubes in which the air respectively gas column is relatively stable. Without much difficulty lye obtained rhythmic precipitation which settled on the glass wall. It has been observed that.such periodicity demands the presence of water vapor for its realization (Rouppert, 1926) and we regard this fact important from our point of view. Since the salt formed is readily soluble and since the wat'er vapor present puts the salt into solution soon after formation, the periodicity is quickly lost. We, however, observed its occurrence under the microscope, and were able to preserve the deposit sometimes by quickly drawing oil up into the tube. (Fig. I I ) . Khen this system is inverted (NH3 into HCI) the formation of periodic rings on the surface of the tube was easily observable microscopically. These were, however, quickly obliterated by extension of crystallization, so that the evidence of periodicity was quickly lost. (Fig. 1 2 ) . Any theory which attempts t'o account for periodic precipitation and fails to explain the above systems is inadequate. In order to do so in accordance with our views w e must assume the presence of water vapor which, as we have seen, must be present if periodicity is to occur. The water vapor present, we believe, makes a temporary compound which can react with the penetrating gas. In forming the initial precipitate (the plug) water will be set free, which is now available for further reaction. Since the internal gas is highly soluble in water, the water set free moves toward the higher concentration of gas, Le. further into the tube unt,il a concentrated solution is formed a t some distance away from the first formed crystals. The concent'rated layer must now diffuse backwards to some point where it will react with the indiffusing external reagent, and a second ring will then be formed. Fluid (TVater):-It is practically less difficult, if still not easy, to obtain periodic precipitation in a fluid medium. The difficulty is overcome by eniploying capillary spaces. We repeated Hedges' experiments of allowing concentrated HCl to diffuse in 30y0 S a C l in capillary tubes. Single crystals were formed periodically and these were large enough to block, but incompletely, the mouth of the tube. Here a t the moment of crystallization water is set free a n d it must diffuse in both directions, diluting the reagents. The
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FRANCIS E. LLOYD AND VLADIMIR MORAVEK
space of too low concentration of the interacting reagent for crystallization must be crossed before another position of high concentration is reached by the entering reagent (Fig. 13a-c). In the immediately foregoing experiments we do not have to consider any reaction product. Such cases are examples of periodic crystallization, the factors which prevent continuous crystallization being water and changes in temperature due to release or uptake of heat of crystallization, complicated by the formation of various crystalline modifications. I n those experiments which follow a reaction product does have to be considered. I n the systems Pb(NO3)2 diffusing into K I and into KaC1 in water in capillary spaces we obtained similar results, the former yielding periodic bands of crystals (Fig. 7 ) , the latter of droplets of metastable compound which later became transformed into crystals. Xotboom (1923) used this method and obtained the same results. A most striking result was obtained with Na2C03diffusing into AgN03 in water in capillary tubes. At first the mouth of the tube was blocked by precipitate. Thereafter marked rings of metastable product attached to the glass wall appeared. I n the course of three days the reaction was completed throughout the tube and we found then that, although the insoluble precipitate occurred scattered everywhere, there was a periodic accumulation of crystals in irregular bands throughout the length of the tubes (Fig. 14a-c). In the system I N BaCl2 diffusing into IK’H2S0, in capillary tubes 0.5 millimeters in diameter, rings of large crystals are formed with fewer small crystals in the interspaces. Doyle and Ryan observe that barium sulphate and silver chloride can be obtained as periodic precipitates only “with great difficulty,” being very slightly soluble. Nevertheless, we have been able to do so in water. W. M. Fischer (1925) used water with a little gelatin and erythrosine admixed, and thought these to be necessary. (Figs. 16,17, IS). Morse and Pierce (1903) got bands of silver chromate in capillary tubes in water, allowing silver nitrate to diffuse into potassium chromate. Gels (Gelatin, Agar and Starch):-In the system .zN Pb(sO3)t diffusing into .IN K I in starch and the same in agar in capillary tubes we obtained very clear cut periodicity of a yellow salt (PbI,), but no such periodicity was obtained when gelatin was used. I n the starch (Fig. 2 2 ) there was a strong tendency toward irregulady, spirals and other abnormalities appearing, or even a discontinuous band cf granules representing the continuous bands elsewhere. Also here, since the salt was strongly absorbed on the glass wall the ring-and-disc structure was pronounced. In the course of time the yellow salt, which has here a metallic lustre, is dissolved or is hydrolyzed, or otherwise changed, and a product is set free-probably 12-which produced a purple coloration in the starch medium, the color being deepest where the yellow salt had occurred. When potato-starch is used instead of the much finer grained corn-starch, no continuous bands are formed. In their place we find a periodicity in the form of discontinuous zones of crystals. These appear even at the very be-
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ginning of diffusion. It is also interesting to note that the release of Izis much more rapid, so that the yellow salt quickly disappears and the starch becomes colored. I n agar beautifully regular and clean cut ring-and-disc bands or spirals occurred in harmonic periodic array, which were dissolved in the entering reagent as the higher concent'rations advanced into the tubes. (Figs. 18, 19). Hatschek (192 I ) and doubtless others (Dogadkin) observed the formation of spirals following the addition of acetic acid to the entering reagent. We have, however, repeatedly found that spiral structures are likely to occur in a t least one in a dozen tubes under the same circumstances, and we have particularly observed that a small bubble attached to the surface of the medium, locally preventing diffusion a t a small point, will induce the irregularity of the first precipitation membrane or surface of the precipitation which will by repetition give rise to a spiral (Figs. 19, 2 2 ) . I n gelatin a t first a yellow, continuous column was formed (Fig. 21). This disappeared as the concentration of the entering reagent increased and the presence of a vitreous, probably organic, precipitate of a colorless salt in the form of globuloids was apparent. As the precipitation advanced, rings appeared independently in the yellow precipitate, which became more and more visible as the yellow precipitate disappeared. These were insoluble in the entering reagent and were composed of an apparently vitreous mass with a cloudy milky-white interior (Fig. zIa). We must here point out that the medium cannot be a simple mechanical framework in these and possibly other cases and we have always to consider the possibility of the gelatin, a t least, entering into the reaction in some way both here and in regard to other systems in gelatin. Indeed recent work done by one of us strongly supports this idea, but the limitations of this account prevents us from exposing our further evidence here. The system AgNO3 diffusing into K2CrZOiwith gelatin, with agar and with starch. (Figs. 20, 2 5 , 26). In these cases there is no evidence that there is other than mechanical action of the medium. Periodicity occurs in all three, but more clearly cut and regular in gelatin and least so in agar. In starch there appears to be no evident protective action so that a precipitate of coarse, dark red crystals appears, the coarser the further away from the mouth of the tube. Here also the periodicity may be absent or may appear in quite clear-cut and evident fashion for a short distance only. Periodicity is most likely in these cases to occur a t low concentrations of theenteringreagent. In gelatin and agar, two salts, red and yellow (AgZCrOJ and a complex between this and K2Cr20irespectively, always appear. In the gelatin both are sharply periodic; in agar only the red is periodic. As the concentration of the entering reagent increases the yellow rings become red and this indicates that the complex is split and that the products proceed in the direction of Ag2Cr04. In agar the yellow salt does not as readily pass into the red salt, the protective action of agar being less than that of gelatin, so that in the former large
1518
FRASCIS E. LLOYD AKD VLADIMIR MORBVEK
crystalline masses build up vague red bands of irregular cloudy form without sharp edges as viewed laterally (Fig. 2 5 ) . The system HgClz diffusing into K I in starch, in agar and in gelatin (Figs. 2 7 - 3 0 ) . I n starch, large red crystals were first formed in a broad band laid down with an evident but not strikingly obvious periodicity followed by a broad band of large, feathery, yellow crystals shot through with long, acicular red crystals with no evident periodicity, these in turn followed by fine red crystals in which a well marked fine periodicity occurred, more evident at greater length. The starch here formed a mere mechanical medium and had only slight or no protective action (Figs. 2 7 , 28). I n agar a more pronounced but still low protective action occurred. Fine red crystals were first formed, but later yellow, in which a fine-grained but definite periodicity occurred (Fig. 29). In gelatin a chemical action on the medium occurs. This shrinks transversely, leaving the glass wall as the concentration of the entering reagent increases; diffusion is much retarded and periodicity does not occur, or is, at any rate, extremely vague (Fig. 30). The system Pb(N03)* diffusing into B2Cr04in starch, in agar and in gelatin. A11 three afforded a very well defined periodicity, Here these media accidently so to speak, have no chemical action and have nearly the same mechanical action toward the'precipitate (Figs, 31, 3 2 , 3 3 , j r , j 2 , 5 7 ) . When the foregoing systems are inverted, no evidence of any periodicity a t any concentration is obtained in agar or gelatin, while in starch a very sharply defined regular periodicity occurs (Fig. 24, I 1 2 , 1 1 3 ) . It is evident that this system is very complicated, since periodicity appears in one direction of diffusion and not in the other. It is noteworthy that starch is here the one medium which affords periodicity for K2Cr04when diffusing into Pb(KOa)2. I t may be a question of diffusion. When the conditions are the same except that dichromate is substituted for chromate, periodicity is obtained in all three media but is better developed in agar than in starch and still better in gelatin. We shall endeavor to offer an explanation of the above in the discussion beyond (Figs. 23, 34, 80). The system 20% NILOH into ZN CoClz in gelatin, for which the gelatin was liquified a t a low temperature ( j o deg.C) a t concentrations of j , 7 and I jyG of gelatin. I n no case was periodicity observed. At the lowest concentration ( 5 % ) of gelatin, a broad band of white precipitate precedes the blue precipitate. In 7y0the white band is not so pronounced and is only slightly apparent in qy0 (Fig. 3 j). If the 2 5% gelatin is liquefied and used a t once for making up the internal complex, bands do not appear as quickly as when the gelatin is kept for 30 seconds at, IOO deg. C. If, on the other hand, exposure to this temperature is sustained for two hours the result becomes complicated by the production of white precipitate and the periodicity of the blue salt is quite irregular (Fig. 36). I t is evident here that gelatin is changed by the treatments indicated and that the resulting changes are reflected in the character of the periodicity.
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In the same system the importance of the concentrations of gelatin is brought out by an experiment in which it varied in concentration from I jyo to 3y0 in eight steps. I n all concentrations the diffusion is constant until periodicity appears, which happens earliest in the highest concentrations when the bands formed hinder the diffusion rate (Fig. 3 7 ) . I n agar (. j to 2 . j%) the same relation holds. Solid M e d i a :--Various observers have obtained periodicity in solid media of divers sorts. We tried gypsum with the system .zS .figKO3 diffusing into .o5K K2Cr207in a capillary tube. The progressive reaction was made apparent by the appearance of small dark red crystals of Ag,Cr04. There \vas evident periodicity, lacking however obvious harmonic arrangement. Summary and Theory
In our previous paper we argued that every reaction can proceed periodically. We now hold that, it can be obtained in every kind of medium if the necessary conditions of space and concentration are supplied. The exceptions are to be found in those cases in which a reagent has reacted with the medium or in which the gel fails to act as a protective colloid for the precipitate. Even t,hen periodicity may occur occasionally. The medium can act chemically or be purely passive in this regard and so act merely mechanically. Viscosity plays a very minor r81e in influencing diffusion. In higher concentrations of the medium, however, more periodicity occurs than in the lolver.
I I I . Efect of Surface. a. The surface of the tube, etc., enclosing the system. We have already indicated in our previous publication the r81e played by the surfaces of the enclosing vessel. The principle is further illustrated by the following additional observations. The system NanCOsdiffusing into iigxO3 in capillary tubes. In t,his reaction coarse crystals are formed which attach themselves on minute irregularities on the inner surface of the tube. These are minute areas of high curvature caused during the blowing of the glass tube, as shown by the fact that the crystals are attached in parallel lines running lengthwise inside the tube. As long as the saturation of the salts resulting from the reaction is too low, scattered crystals only occur. When the saturation is sufficiently high, a ring of separate lines of crowded crystals is formed, the whole picture showing periodicity. I n this case the reaction product is very strongly positively adsorbed on the glass wall. If this were not the case the precipitate would be distributed as a diffuse, vague cloud of particles in the corresponding zones (Fig. 14a-c). The system KsCrOr diffusing into Agn’Os. I n this case we give one in which the concentration of the reaction product is too low to supply sufficient material to form a complete band. Since here the precipitate is very strongly adsorbed on the glass wall, it attaches itself to the minutest irregularities in
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FRANCIS E. LLOYD AND VLADIMIR MORAVEK
periodic fashion. Since the irregularities of the same order of magnitude occur in a particular line, the clumps of precipitate are t o be found along this line, of two or more. If the precipitate is not positively adsorbed it will be found scattered throughout the whole medium and no periodicity might then be observed (Figs. 38, 39). I n the case in which oversaturation occurred, as in the system 2 % KZCr207 diffusing into .5N AgN03 in gelatin, the reaction product, being strongly positively adsorbed on the glass wall, is deposited in a dense ring which so far depletes the corresponding transverse zone that a reaction of oversaturation is reached only in advance more or less of the already deposited ring. Thus is formed a disc corresponding to the ring (Text Fig. xb.) This is the “Saturn
TEXTFIGS. Ia ANI xb
structure,” so called by Popp. We must accordingly always find the disc to be laid down somewhat later than the corresponding ring and this, more easily in the greater depths of the tube, can be seen to occur, the ring being formed some time before the disc. The time interval increases the deeper in the tube we go. Between the rings the precipitate is formed more slowly, due to the low saturation as the entering reagent advances and it is deposited on the glass wall. If in sufficient amount it may entirely obscure the periodicity (Figs. 40, 41,44-46,77). Since adsorption will play a greater r61e in small tubes, it becomes less important in large tubes in which the surface therefore will play a minor rdle. The deposit of precipitate will go at once to the disc and the ring is not formed appreciably earlier. In order to explain the ring-and-disc structure Dogadkin has postulated a curved surface (meniscus) of the medium where the external reagent enters. I n order to assure this structure, the meniscus must be concave. This view is quite without foundation, as we have been able to obtain the ring-and-disc structure when the meniscus in question was curved in the opposite sense. It is, furthermore, absent when the precipitate formed is negatively adsorbed on the glass wall.
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We tried to determine if the kinds of material would have any effect, using the system 20% K”,OH diffusing into 2N CoC126H20 in IO% gelatin in tubes I O millimeters in diameter of the following materials: silica, soft glass, hard Jena glass, chemical glass tubes lined with caoutchouc, collodium, cacao butter, paraffin and lecithin. I n all, the rates of diffusion and the number of rings were the same (Fig. 47).
Summary: The surface plays an important r8le in capillary tubes, but a less important or even negligible r81e in tubes of larger diameter. Accordingly we may obtain periodicity in the former, but not in the latter. The relative importance will depend on the nature of the precipitate or on the degree of adsorption. Wulff (1929) had investigated the deposition figures of iodine in vacuo and found the first deposition always in places where there are minute curvatures, scratches and tension lines on the glass surface. We have observed the same and have offered evidence in many of our illustrations. Such minute irregularities as Wulff speaks of can account for many appearances in the case of substances adsorbed on the glass wall unexplained otherwise. b. Surfaces in the medium, inhomogeneity. The following interesting behavior took place in the system .zgN AgNOS diffusing into . 2 jN S a c 1 in gelatin in test tubes. The preparation which was observed during two months, yielded at first a very fine periodicity observable only through the microscope and later broad bands with no interspaces appreciable to the eye, so that the bands could not bedistinguisheduntilthepreparation was exposed to the light, when a differential blackening, due to difference in densities, occurred. The preparation had been kept in the dark. During the period, fungus hyphae developed in the column of gelatin beyond the prccipitate and when these were overtaken by the reacting salt a microscopically coarse crystalline precipitate was deposited (AgC1), which, being negatively adsorbed on the glass wall, was scattered throughout the medium. It was, however, positively adsorbed on the fungus hyphae so that the form of the fungus was made evident by the crystals attached to it. (Fig. 48). Appearances in the system h g N 0 3 diffusing into K2Cr207in gelatin when the experiment is carried out in the classical fashion of Liesegang, i.e. when a drop of the entering reagent is placed on a field of K2Cr20i-gelatin in a glass plate (preferably a Petri dish so that it may be protected from evaporation the more easily). Vnder these conditions three sets of rings (here well enough so called) are produced, not two only as usually supposed. First a set of fine colorless rings (regarded as phosphates by us). These are produced with great regularity and uniformity until a t some distance from the center of diffusion they lose all regularity and periodicity is quite lost. On the colloidal suspension forming the precipitate of these rings, a second dark red silver chromate set is formed by adsorption but the regularity of these is disturbed by the loss of regularity of the primary rings and more or less by the presence of various foreign bodies (minute fibers, particles, etc., etc.) in the gelatin. The rings of the third set are brown and ribbon-like, occurring only at the
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F R A S C I S E. LLOYD AND VLADIMIR MORAVEK
gelatin-air interface and while obviously related to the second set, to some extent are independent. Such rings do not occur a t the gelatin-glass interface and are evidently circumstanced by the presence of the gelatin-air interface, which does not occur in tubes. If a sheet of thin glass is placed on the gelatin so that the radially extending diffusion must pass partly under the glass, this system of rings is formed only at the gelatin-air interface up to the edge of the glass cover, beneath which it is not to be found, though there is some scattered granular material adsorbed at the gelatin-glass interface, due especially to dirt, etc. on the glass. It becomes apparent that we are here dealing with the saturn structure which occurs only as above described, viz. when a gelatin-air interface is available. The ribbon-shaped ring can occur, if the layer of gelatin is thin, without any accompaniment of precipitate inside the gelatin, just as the ring alone may occur in a test tube beyond the rings-and-discs. In our previous paper we have referred to similar conditions procured experimentally. Fungi, of course, grow readily on some media. In . z 5 Sa?11P04diffusing into . 5 to .006K CaCl? in test tubes, the preparations of which were observed during the period of a month, there occurred an abundance of fungus colonies which grew in spherical form. Since in this case the reaction product was negatively adsorbed on the fungus, the dense spherical colonies merely formed barriers which hindered the diffusion of the reagents, more or less, according to the position of the colony with regard to the band being formed. The forms of the precipitate were therefore determined by the new directions of diffusion, affording curious funnel- or dome-shaped irregularities in the disc of the ringand-disc, which latter as a hole is referable to the positive adsorption on the glass wall (Figs. 4 2 , 43, also text Fig. Ia). K i t h the system Pb(XOsjs diffusing into K2Cr0, (.gSinto . I to .0031?rT) in agar in capillary tubes we obtained quite unusual and a t present inexplicable results. For the most part the resulting periodicity consisted in fine regular bands of very fine precipitate not adsorbed on the glass wall. I n one tube of the highest concentration of entering reagent two periodic systems became visible (a) a relatively coarse band, each band preceded by an interspace relatively free from precipitate and (b) fine banding locally confined to small columns of the medium, being absent elsewhere. I n a transverse section we found that there was a very irregular distribution of precipitate, rather obviously connected with a considerable inhomogeneity in the material of the medium. A section from another tube in which the fine bands were uniform and spanned the whole lumen of the tube, showed a quite different picture, only very minute irregularities appearing (Figs. 49, j o , j ~ 5 ,2 ) . I t is much more likely that starch should produce such irregularities if prepared so that the grains are incompletely hydrated, when nuclei poor in water will occur suspended in a colloidal mass of more highly hydrated starch. Systems especially in which there is a delicat,e equilibrium between periodicity and non-periodicity are easily upset in starch while in agar and
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gelatin they afford very regular pictures. This is especially well shown in the system Pb(N03)*diffusing into KI in corn starch paste (Figs. 19, 2 2 ) . \Then potato-starch (Fig. 2 2 , three right hand tubes), with its much larger grains, is used, while periodicity is produced, especially in finer capillaries (.2-4. mm. diameter) the precipitate consists wholly of coarse crystals and there occur no continuous bands such as we see in the five left hand tubes of Fig. 2 2 . By introducing certain adjuvants to the medium we have changed the surface tension of the minute components (micellae, granules) of the medium and we have sought to determine the effect that such additions would have on the periodicity of the system when substances which lower the surface tension were introduced. The following substances were used and the corresponding surface tensions at the air-water interface as determined by means of a de S o u y tensiometer are given as follow: Sodium oleate do. Capryl alcohol saturated solution . I do. do. . 3 Cholesterol by the Aloravek method .03 do. do. 003 do. do. 1%
.
17'
surface tension 3 7 . 3 41.1 ** * 33.3
56.9 42.2
4i.8
not measured.
These were added to CoC12 in 10% gelatin. In sodium oleate and cholesterol the banding was identical with that in distilled water, the control. In capryl alcohol in both solutions there was a considerable departure from the control in the great prominence of white precipitate with few and diffuse blue rings. \\-e must assume a chemical interaction in the case of the capryl alcohol, for it is clear that the changes in surface tension had no effect (Fig. j 3 ) . Macro-inhomogeneities, as in starch and agar (especially when prepared by partial hydration), affect the behavior of the reagents and of the precipitate in any system.
I I-. Final F o r T of the Precipitate. Crystalline precipitate. The precipitate occurs in the form of crystals (micro and macro) in water and in media which do not act protectively and which therefore behave, aside from purely mechanical support, as if not present; e.g. zyo CaC12 diffusing into zyo I C O O S H , ) ~in gelatin in capillary tubes. Here no trace of periodicity was visible. The crystals were small placques, single or aggregated into small spherocrystalline groups. In the inverse of this system we obtained a t first small crystals which became larger the further in the tube where the diffusion was slower. A very slight indication of periodicity was evident in one or two tubes e.g. the right hand tube, Fig. 55. (Figs. 54, s ; , 7 2 ) . The precipitate may be a t first diffuse and suddenly pass into a periodic condition if the entering reagent is hindered from rapid diffusion as e.g. by a
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FRANCIS E. LLOYD AND VLADIMIR YORAVEK
bubble (Fig. 57). Whereas in the system K2Cr207diffusing into AgK03 in starch (.5K into . I to .0031N) we observed all intergraduation between periodicity and nonperiodicity (Fig. j6). I n the system Pb(N03)2into NaCl in gelatin (.5K to . z 5 into . j to .zjN) we obtained periodic bands of spheroidal crystals which very slowly developed from the already laid down colloidal precipitate (Figs, 58, 59). I t is evident that periodicity is not a t all conditioned by the colloidal state of the precipitate as we obtained well defined periodicity in many experiments in which the salts formed are hardly to be obtained in colloidal form, as in the case of BaS04 in water (Figs. 15, 17). Colloidal precipitations: growth forms. In the systems KZCrz0,diffusing into AgKOs in starch (.5X into . I to .003 IN) we obtained the reaction product in the form of semi-permeable cells, more or less in periodic grouping. We recognized these as Traube membranes, which grew in size after breaking out and producing various curious and fantastic shapes (comma, comet and other), We removed some of them, mounting them for microscopic examination in the entering reagent, when on crushing we could observe the formation of new membranes and the growth of older ones (Figs. 60-64). Similar, very uniform, comet-shaped membranes were obtained in 50% AgNOa diffusing into .jS K2Cr20iin gelatin in a glass plate. The thinness of the gelatin was here a factor (Fig. 60, inset). Sharp banding. The reaction product according to rate of diffusion and of concentration yielded different forms of colloidal precipitate in the two main types of diffuse and sharp (Figs. 6 j , 67, 68). These appeared, e.g. in the systems (?jH,)zHOP4 diffusing into CaClz in gelatin (.zN into .I to .006K) and KH40H diffusing into MgCll in gelatin, as also in some other systems. Popp (1925) obtained bands in the system ammonia and magnesium chloride in both directions, whereas we obtained bands usually only when the ammonia was the entering reagent. Once, however, we got the same result as Popp when 0.5 sat. s o h . MgC1 diffused into ISSHaOH in gelatin. (Fig. za, Plate IX) This does not, however, militate against our view. We used various concentrations of gelatin and our results were the same as hers, so far as they go. In 474 Pb(KOa)2into 470 Bi in gelatin we obtained a yellow colloidal cloudy periodic precipitate which was changed on the increase of concentration of the entering reagent to white granules which grew into spheroidal crystals grouped periodically (Fig. 66). There is difficulty in the interpretation of Pb and Ag reactions in gelatin. As is well known, Pb (NO3)Z with gelatin alone afford a periodic precipitate negatively adsorbed on the glass wall. If tubes are thus set up as control, similar tubes with KI give, in addition, a cloudy colloidal yellow precipitate with a sharply Danded white periodic precipitate within the yellow. We have already pointed out that when the precipitate is colloidal and positively adsorbed on the glass wall (or other suitable interface) the ring-anddisc structure always appears. Whether it is well defined and regular or ill defined and irregular or otherwise depends on secondary conditions, especially
FURTHER STUDIES IN PERIODIC PRECIPITATIOS
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on diffusion rates. Bradford’s observations (1919) on the adsorption of a cloudy or colored precipitate in the region of the band accords with ours but does not militate against our theory. Periodicity is evidently independent of all possible states in which the reaction product may occur. The problem of periodicity then is to be solved quite in disregard of these conditions, contrary to some theories which have been advanced.
Influence of one precipitate o n a second. Phosphates i n the gelatin. System A g S 0 3 diffusing into S a n H P O aand inverse (Figs. 71, 73, 86). When we experimented with these we found in all cases two systems of rings, one of fine bands in advance and, adsorbed on these, a second dark appearing precipitate. ]Ye attribute these fine rings tentatively to phosphates in the gelatin, chlorine being absent. They are developed most abundantly and uniformly in the lower concentrations of the internal reagent. In the higher concentrations the fine rings were more poorly developed and coarse spheroidal niasses showing weak or no Periodicity took their place. In certain intermediate concentrations we obtained intergrading conditions, in which the coarse precipitate was adsorbed on the finer. In experiments in which AgSOs is allowed to diffuse into gelatin without additional reagent a fine periodic precipitation is obtained and this, when other reagents are added, forms a groundwork for the additional precipitat,e to ‘he adsorbed upon. Stansfieltl (19 I 7 ) also obperved n niicroscopical periodicity in the system silver nitrate diffusing into Iyas the chromate. This groundwork again seems t o be clue to phosphates in the gelatin. When S a C l is added t o the gelatin :i tlifferent and much finer periodic precipitation is obtained with readiness recogniz bly different from phosphate rings which are more soluble in the entering reagent than the chloride. Liesegang examined the behavior of silvcr nitrate diffusing into potassium chromate-gelatin and the inverse, observing the fine white bands which he at,tributed to chlorides, which, however, he stated ns not being necessary to the formation of the chromate bands and as not present when the chromate diffuses into the gelatin. S o r are they present when the medium is prepared by adding h g S O s to the gelatin, followed by K2CrzOi, (and therefor containing the reaction product, but neither free chlorides or phosphates). The excess K2Clr?O7reacts with the entering .IgSOa periodically, but no fine banding is seen (Fig. 7 I a ) . Lagergren (1899) observed negative adsorption of NaCl and Bradford on the addition of this to ILCr20i-gelatin obtained only diffuse cloudiness with AgXO3, whereas we have found clear cut periodicity. K e agree to some extent with Bradford that there is something either already present in the gelatin (impurities, he calls them) or, as we have already suggested, which appears in the course of the reaction periodically as fine bands facilitating the periodicity of the chromates of d g and Pb, but which assuredly is not a sine qua n o u . If mineral impurities are present we argue that they are phosphates since we obtain similar periodicity when Ag or P b diffuses into
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FRANCIS E. LLOYD AND VLADIYIR SlORAVEX
gelatin containing phosphate (Figs. 70, 7 1 , 74, 75). On the other hand we admit that the fine bands may be due to a reaction of Ag or P b with the gelatin itself. When Pb(FO3)2 diffuses into gelatin alone a mixed cloudy and coarse periodic precipitation is formed, the latter composed of spheroids of pearly appearance. When K2Cr0., is added to the gelatin the chromates, being weakly developed at low concentrations of the internal reagent, form insufficient surface for the adsorption of the phosphates and these are then present as scattered globulites. I n the higher concentrations of chromates the very numerous bands present allow the full adsorption of the phosphates, which are then periodically deposited thereon (Fig. 33). It should be added as confirmatory of the foregoing statements that we never obtained t'he precipitation of phosphates in agar alone or in starch alone. Also we obtained with the system P b ( S 0 3 ) 2diffusing into K2Cr04in agar the same fine bands, which we attribute to chromates, as we did in the above mentioned experiment in which such bands serve as a groundwork for the emplacement of the coarser phosphate precipitation (Fig. 5 7 ) . Summary: There can be at least two sets of periodic precipitation in a single system. When one is adsorbed positively on the other the appearances are complicated and are different according to the concentrations of the reaction products.
V . InJluence of Concentratiom. (a) Silt'er chromates. System I
