(From the Sir William Ramsay Laboratories of Inorganic and Physical Chemistry, University College, London)
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1 DISTRIBUTION OF HYDROCHLORIC ACID IN GELATINE GELS BY L. HALPERN (From the Sir William Ramsay Laboratories of Inorganic an Physical Chemistry, University College, Lonon) (Accepte for publication, March 16, 1931) In a recent series of publications (1), E. J. Bigwoo has observe the presence of a permanent concentration graient of hyroxyl ion when, uner suitable conitions, ilute aqueous soium hyroxie iffuses into a gelatine gel. We sought to reprouce similar concentration graients an to measure them quantitatively in orer to fin conitions uner which we coul test the theoretical expressions erive by F. G. Donnan (2) for such a system. This theory presupposes a graient of protein micelle concentration, naturally or artificially forme. We thought that the phenomenon escribe by Bigwoo ought to satisfy these conitions but our attempts prouce no system that coul not, in our opinion, be satisfactorily explaine by the laws of simple iffusion. There was nothing to inicate the presence of a protein concentration graient. In our experiments Coignet's Gol Label gelatine was use, purifie by electrolysis (3) (ash content per cent), an containing traces of thymol. 9 cc. columns of 3 per cent, 5 per cent, an 8 per cent gels containing equal amounts of brom-phenol blue were cast in test-tubes an covere with 5 cc. "0.015 N HC1 an 1 cc. of toluene. The tubes were then seale an kept in the refrigerator. A control tube containing water instea of the aci showe no color change in the inicator throughout the experiment. The istance iffuse,, was measure as cc. from the top of the gel to the bounary of the inicator change. After about the fifteenth ay reaings become less sharply efine because of the iffuseness of the zone of color change. From Fig. 1 it will be seen that ~-, which woul be a constant in orinary iffusion, is here a ecreasing quantity. This might suggest an approach to a stable concentration graient within the gel at equilibrium. 575 The Journal of General Physiology
2 576 HYDROCHLORIC ACID IN GELATINE GELS However, in 3 weeks the inicator in the 3 per cent gel was yellow throughout, in 2½, months the color of the 5 per cent gel was uniform throughout but correspone to a ph of about 4, i.e., within the range of the inicator. The 8 per cent gel still ha a yellow, very iffuse zone a t the top. We repeate these experiments using 0.01 N HC1 whose concentration was kept constant. For this purpose a gently flowing reservoir C o 0300 t (~ ~ O 3O0 Time FIG N HC1 in limite amount an constant level siphon system was use. It will now be observe that ~-~ remains constant within experimental error (Fig. 2). Sobotka an Sabin (4) have also foun that the constant gives lower values at the beginning of the experiment. If an equilibrium were being approache, one woul expect ~tt to ecrease continuously. The
3 L. HALPERN 577 ecreasing values for the first experiments can, however, be explaine as in simple iffusion by the exhaustion of the iffusing substance. Much more precise forms of the equation ~tt = k have been formu- late by Aair, Stiles, an others (5), but it was not thought necessalt to use them here. It will thus be seen that all the conitions impose in Bigwoo's experiments woul lea one to expect an extreme case of slow iffusion which might be easily mistaken, with relatively crue measuring ool ~ c 37. gel O C r I I.I,,, I I I I, ~ ~ TLme FIG N HC] at constant concentration methos, for a stationary system. His rather concentrate gels (8 per cent) were in contact with a limite amount of very ilute alkali (usually less than N) whose ph, as he shows, was continuously ecreasing. No mention was mae of a preliminary purification of the gelatine which orinarily contains 2-3 per cent ash. The active concentration of the alkali is further ecrease by combination with the gelatine.* Sobotka an Sabin (4) have emonstrate the influence of this factor on spee of iffusion. Using the average value for the iffusion constant foun in these experiments for an 8 per cent gel * I cannot agree with Bigwoo's conclusion that the gelatine in an 8 per cent gel is a negligible source of neutralisation for iffusing OH- ions when the ph of the outsie solution is equal to or greater than 10 (Reference lc, p. 711).
4 578 HYDROCHLORIC ACID IN GELATINE GELS in contact with continuously renewe 0.01 N HC1, calculations show that it woul take 3 ½ months for the inicator to change throughout the 9 cm. column an that towar the en the bounary, if clear, woul take 2 weeks to move 0.2 cm. It is not remarkable, then, that this shoul appear to be a system in stationary equilibrium. The fact that ilute aci was use in these experiments instea of alkali shoul not affect the results as long as the gels were not, at the en, at the isoelectric point but within the zone of color change of the chosen inicator. In reference to his more recent experiments Bigwoo points to the shifting of ion concentration, ue perhaps to the liberation of aci valency in proportion to the evolution of the gel structure. In our A B I Time Fzc. 3 C opinion, this reversal of the various ph niveaux after a time can be explaine by an examination of the orinary form of iffusion curves for ifferent concentrations (Fig. 3). If the concentration of iffusing substance were orginally A an graually roppe to B, the concentration at any one point in the gel woul return to the value for the curve for B at that time. At a certain time this ifference in concentration woul mean a return to the original color of the inicator. In any case, a reversal of the zone of color change after replacing the supernatant solution with water oes not prove that a slow iffusion ha not been taking place towar the interior of the gel. It may be a question of simply varying the relative intensity of iffusion in each irection.
5 L. ~A~PEaN 579 The theory of the existence of a permanent concentration graient of hyrogen ion is base on the assumption of a permanent graient for protein micelles. Light-scattering experiments, carrie on by an with the kin assistance of Dr. K. Krishnamurti in these laboratories, o not inicate such a graient uner the given conitions. When he allowe uniform gelatin gels to imbibe small amounts of istille water an measure the light-scattering power at various heights in the column, he foun that it became ientical throughout if sufficient time were allowe. Similarly, he measure 5 cm. columns of 3 per cent isoelectric gelatine in contact with 8.5 cc ~ HC1. The change in turbiity was the same throughout the gel in 2 weeks. The greater concentration of aci use in this investigation allowe equilibrium to be reache in a shorter time but the metho is sensitive enough to etect any appreciable graient (6). Since we were unconvince that permanent concentration graients coul be obtaine in this way, we prepare one by layering equal amounts of gelatin sols of varying concentrations upon each other in a test-tube, being careful to avoi air-gaps or sharp interfaces within the gel. There was a uniform concentration of brom-phenol blue in the column which was then covere with 10 cc ~ HC1. This was renewe in 4 ays an remove in 11 ays. After 41 months a color graient still persiste within the gel, the hyrogen ion concentration not having istribute itself evenly as ha been the case with uniform gels uner similar conitions. An attempt was mae to test the conclusion that, with a protein concentration graient an a iffusing electrolyte, the concentration of each ion woul also be a graient but in opposite irections. This is expresse by Donnan (2) in the following ifferential equations: an h ch p x cp x 2(1+ bh) + cp l+bh A-) p,~(a-) = -- c p :~ 2(I + bh) + cp q- l +bh
6 580 HYDROCHLORIC ACID IN GELATINE GELS where h an A- are the concentrations of the free cations an anions respectively of the ae electrolyte at place x in the gel, c an b are constants in the simple Langmuir equation for the reversible asorption of cations, an p is the micelle concentration. Therefore, if p > O, then h < 0 an [a-i > O. x x x After 4½ months the gel was remove by cracking the tube, ivie into sections, an analyze. The inicator showe a ph increasing TABLE I No. Weight of gel Concentration gelatine Cl- (total) CI- per gin. gel gm gin. per cent 1,89 rag rng , The last column is correcte for the small blank on the gelatin. The chlories were estimate by a metho similar to that given by Van Slyke, D. D., J. Biol. Chem., 1923, 58~ 52. graually from Sections 1 to 9. Table I gives the results of analysis. Of course, a graient of protein concentration oes not necessarily mean a graient of micelle concentration but it is unnecessary to repeat Here the simplifying assumptions that were use as the basis an limitations of Donnan's theory. It will be seen that the conclusions rawn by Donnan are verifie
7 T. I~A~I~EI~lq 581 in a general way, since the anion (C1-) concentration varies in the same irection as the protein concentration, while the cation (H +) varies in the opposite irection. The author wishes to express her thanks an appreciation to Professor F. G. Donnan for the suggestion of the problem an for his constant interest an avice uring the work represente by this paper. BIBLIOGRAPHY 1. Bigwoo, E. J., (a) Am. Y. Physiol., Proceeings, Oct., 1929, 90, 285. (b) Compt. ren. Soc. biol., 1926, 96, 131,136, 199; 1929, 102, 322, 324, 600, 602; 1929, 103, 45, 47. (c) Tr. Faraay Soc., 1930, 26, 704. (Appeare since the completion of this work.) 2. Donnan, F. G., Kolloi-Z., 1930, 51, Knaggs, J., Manning, A. B., an Schryver, S. B., Biocttem. J., 1923, 17, Sobotka, H., an Sabin, A. B., J. Am. Chem. Soc., 1928, 50, Aair, G. S., Biockem. Y., 1920, 14, 762. Stiles, W., Biochem. J., 1920, 14, 58. Stiles, W., Proe. Roy. Soc., Lonon, Series A, 1923, 103, 260. Mann, C. E. T., Proc. Roy. Soc., Lonon, Series A, 1924, 105, 270. Watanabe, M., Kolloi-Z., 1923, 32, Krishnamurti, K., Proc. Roy. Soc., Lonon, Series A, 1929, 122, 76.
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