THE COL4GULA4TIOSOF A4LBt-lIIS B T ELECTROLYTES BY TI'ILDER
D. B-AA-CROFT
The recent work on albumin has been t o the effect that definite compounds are formed with acids and bases In so far as albumin is present as a second phase. conclusions based on conductix-it>- measurements are subject to serious error, and conclusions based on electrometric measurements may be in error In so far as albumin is present as a second phase, adsorption phenomena must play some part It seems desirable therefore to see to what ehtent the coagulation of albumin b!. electrolJ-tes can be accounted for uithout postulating the formation of definite compounds. It will then he possible t o see to what extent one must postulate the existence of compounds. I n other words, we nil1 give ups for the time being. the dogma that albumin is an amphoteric electrolyte. 1T.e start with the assumption that a slightly acid or slightl!- alkaline solution of albumin is a tnwphase colloidal sJ-stem which is least stable whei; electrically neutral. TT-hen the albumin adsorbs the anion relati1 el? more than the cation, as in alkaline solutions, it becomes charged negatively and consequently moves to the anode under electricsl stress V'hen it adsorbs the cation more than the anion. as in acid solutions, it becomes charged positil-ely and consequently moves t o the cathode under electrical stress If vie take a negatively charged albumin, in neutral or slightly alkaline solution, and add a salt solution, the effect of an adsorption of the anion will simply be t o increase the negatiye charge and consequently t o make the colloidal solution more stable. The more strongly adsorbed the anion is, the less readily will the albumin be precipitated by cations. On the other hand, the adsorption of a cation decreases the electrical charge and con~
- ~ _ _
Pauli a n d Hir,chEeld
Blochem
&it,
62, z+j 19141
W i l d e r D . Bancrojt
350
sequently makes the albumin less stable. The more readily adsorbed the cation, the lower will be the concentration a t which it will cause precipitation. In any given case we shall therefore be able to arrange the cations and the anions in series, depending on the way in which they precipitate albumin. It must be remembered that in many experiments with white of egg, the globulin has not been removedI1 in which case the data refer to the globulin rather than to the albumin. In Table I are some data by Hofmeister? for a neutral or slightly alkaline solution. The solution contained 0 . 2 g albumin per I O cc; the salt concentrations are given in gram equivalents per liter; the temperature was 30’-40”; the numbers in parenthesis give the valences of the anions:
TABLE I Concentration in gram equivalents per liter K
SHa
Ka
~~
Tartrate (2) Sulphate (2) Phosphate (2) Citrate (3) Acetate (1) Chromate (2) Chloride (1’ Nitrate (1) Chlorate (1) Iodide (1) Sulphocyanate ( I
I
.5I -
I .j6
2.72
.60 I .6j I .68 I .69 2.61 3.63
2.03 2.51 2.71
I
.61 1.67 I .6j 2.67 3 ’ 53 I
-
-
-
5.42
-
-
j.j 2
S o precipitate in saturated solutions t
< L
,(
L
The ammonium salts precipitate at distinctly higher concentrations than the sodium and potassium salts and therefore, by definition, are adsorbed less than these. There is not much difference between the sodium and potassium salts, and it is probably wiser t o attribute what little difference there is to experimental error, at least temporarily. This is the more advisable because Pauli3 considers that potassium 2
3
Pauli: Pfliiger’s Archiv., 78, 31j (1499). Pfliiger’s Archiv., 24, 2 4 7 (1887). Hofmeister’s Beitrage zur chem. Physiol., 3,
2zj
(1903).
Coagzdation
sf Albumi?z bj'
Elertrolj'tes
351
salts are less effective than sodium salts, whereas Hofmeister's data point t o their being somewhat more effective. Since Hofmeister found that lithium sulphate precipitated albumin a t an equivalent concentration of I j q , we conclude that lithium salts are adsorbed slightly more than sodium and potassium salts. Magnesium sulphate precipitates at 2 65, which seems to imply that magnesium salts are adsorbed less than ammonium salts. V'hether we draw this conclusion or not depends on whether we take the equivalent concentration or the ion concentration as the standard. For equivalent concentrations of sodium sulphate and magnesium sulphate the precipitating concentrations are I 60 and 2 6 j . The gram molecular concentrations of sodium and magnesium ions are I 60 and I 3 j , respectively, assuming complete dissociation. On this basis magnesium ions precipitate a t a lower concentration than sodium ions. If we do not postulate complete dissociation, the effect is even more striking because magnesium sulphate is dissociated less than sodium sulphate. It seems to me that the ion concentration is the important one because it is the ion which is adsorbed. Consequently I deduce that the order of precipitation and of adsorption is l l g > Li > S a , K > NH4 when referred to ion concentrations, magnesium ions being adsorbed the most and ammonium ions the least. This point has been overlooked entirely so far as I know, all people putting the precipitating power of magnesium salts below that of ammonium salts. The present way of looking a t the matter seems t o be more sound theoretically, and it has the advantage of bringing the magnesium salts more nearly in line with the salts of the other bivalent metals. If we deduce the order of adsorption of the anions from the data in Table I we get : sulphocyanate, iodide > chlorate > nitrate > chloride > chromate > acetate, citrate > phosphate > sulphate > tartrate, the sulphocyanate ion being adsorbed the most and the tartrate ion the least. Pauli' finds that the 1
Hofmeister's Beitrage zur chem. Physiol., j, 243 1903)
W i l d e r D . Bazzcrqft
352
tartrate comes in between the acetate and citrate. If so, Hofmeister's determinations with the sodium and potassium tartrates must be in error. Freundlich' considers that the concentrations should be given in gram molecular weights per liter instead of gram equivalents in which case one gets Table 11: TABLE I1 Concentration in gram molecules per liter ~~
~
Citrate Tartrate Sulphate Phosphate Chromate Acetate Chloride Nitrate Chlorate Iodide Sulphocyanate
~
~~
K
Sa
o j6
o j6 o 78 o 80 o 82 1 30
0
75 -
0
80
33 67 3 53 1 I
I 69 3 63 5 32 5 52
-
__
"4
''
0
90
o 91 I or I
2 j -
'
-
-
KO precipitation in saturated solutions ,L chlorate > nitrate > chloride > acetate > chromate > phosphate > sulphate > tartrate > citrate, the sulphocyanate ion being adsorbed the most and the citrate ion the least. Since we are agreed that the ion concentration is what counts, this seems at first the more rational way of arranging the data for a study of the anions. It has the further advantage that ammonium tartrate fits in as it should, whereas it was abnormal in Table I. On the other hand there is the difficulty when comparing chromate with acetate for instance, that the concentration of sodium or potassium as ion is double in the gram molecular solution of chromate what it is in the acetate solution. Since the albumin is negatively charged, it is precipitated by cations and consequently the error introduced in Table I1 by doubling the concentration of the cation, may be more serious than the error introduced in Table I Kapillarchemie, 42 j (1909).
Coagulation
OJ
.4lbailniiz by Electrol2'tes
353
by an inaccurate formulation of the anion concentration. There was a similar error for the cations when comparing ,IlgS04 with NaeSOl 2 , but the error due to the change in concentration of the sulphate is relatively small, because this anion is not adsorbed to any great extent. With the anions the case is different and it seems to me that the evidence is not sufficient to enable us to decide conclusively between Tables I and 11. It will therefore be safer for the present to consider only those anions which come in the same order in both tables. The order of these anions is: sulphocyanate, iodide > chlorate > nitrate > chloride > acetate >phosphate> sulphate > tartrate, the sulphocyanate ion being adsorbed the most and the tartrate ion the least. These deductions as to the order of adsorption of the cations and anions are made specifically to fit the facts and are therefore worthless unless confirmed independently. JVe can get the needed independent confirmation by considering the behavior of albumin in slightly acid solution. Here the more strongly adsorbed cation will increase the electrical charge and will consequently make the albumin more stable, while the more strongly adsorbed anion will cut down the electrical charge the most and will precipitate the albumin the most readily. With an acid concentration of o 0 1 - 0 . 2 LY HC1 Pauli' found that the order of anions and of cations reversed, the order for the anions becoming : sulphocyanate >iodide >bromide > nitrate>chloride>acetate. This is as it should be, and the same relation holds with a stronger acid, o 03 AYHC1. The behavior of the cations becomes abnormal, however, in the 0 . 0 3 S HCI solution, the ammonium salts precipitating a t a higher concentration than the corresponding sodium salts, just as they did in the slightly alkaline solutions. I do not pretend to account for this unexpected shift. The most that can be done a t present is to formulate the general principles. A careful experimental study of each case will be necessary Hofmeister's Beitrage zur chem. Physiol., 5 ,
27
(1904).
W i l d e r D . Bawrojt
354
before one can be certain what is the cause of special alleged discrepancies. Copper and zinc salts precipitate negatively charged albumin even at concentrations of -11 1000 or less, from which we conclude that these cations are adsorbed very markedly. From what we have seen in regard to sodium and potassium salts, we should expect that a higher concentration of zinc iodide would be necessary to coagulate albumin than if one added zinc sulphate. The experiment has not been tried in just this form so far as I know: but Pauli’ found that addition of sodium iodide or sulphocyanate cut down the precipitation when a very dilute zinc sulphate (0 005 S)was used. The order of anions was chloride > nitrate >bromide >iodide > sulphocyanate, the most precipitate occurring with the chloride and the least with the sulphocyanate. Curiously enough, it was found that iodide kept the solution clear when 0 . 0 2 S %SO4 was used, while sulphocyanate caused a heavy precipitate. With 0 . I S ZnS04 the addition of sodium salts caused a precipitate decreasing in the order : sulphocyanate > iodide >nitrate > chloride > sulphate. This is what one would expect if one had a positively charged albumin instead of a negatively charged one. Unfortunately this point was not thought of by Pauli. I am inclined to believe that this reversal of the anions is connected with the fact that zinc sulphate does not precipitate albumin at all when present in normal to double-normal concentrations. If we start with a negatively charged albumin and add acid, we neutralize the negative charge and precipitate the albumin. On adding more acid we get the albumin charged positively and peptonized. There is no reason to suppose that this action is confined to the hydrogen cation, and I believe that albumin which is not precipitated by a gram molecular solution of zinc sulphate will be found to be charged positively. In that case the order of the anions would be reversed, as Pauli found. YiJith lower concentrations of zinc 1
Hofmeister’s Beitrage zur chem. Physiol., 6 , 233 (1905).
Coagulation oj Albumi?z by Electrolytes
355
sulphate we should expect the reversal to occur first with the sulphocyanate which is exactly what happened. Szilardl finds that thorium and uranyl nitrates peptonize albumin, which is what they should do in case these cations are adsorbed very strongly. If we start with an acidified solution the addition of zinc sulphate or copper sulphate should have very little effect. Werigo? reports that copper sulphate does not precipitate albumin in presence of hydrochloric acid. Unpublished experiments by Mr. H. I. Cole showed that o 2 cc -11 I O ZnSOi produced a distinct cloudiness when added a t 30°-400 to I O cc of a slightly alkaline albumin solution (containing globulin). n’hen the solution was acidified it was necessary to add more than I O cc ?I1 I O ZnSOi to produce the same cloudiness. 3fr. Cole also confirmed Pauli’s results that addition of potassium iodide to a slightly alkaline solution of albumin cut down the precipitation by zinc sulphate. He also found that in acidified solutions potassium iodide precipitated at much lower concentrations than sodium chloride. Pauli’s results with calcium, strontium, and barium salts3 confirm the hypothesis put forward to account for the effect produced by zinc sulphate. He found that with these salts the order of the anion reverses, and becomes: sulphocyanate > iodide > bromide > nitrate >chloride > acetate, the sulphocyanate causing the most precipitation and the acetate the least. If we assume that the calcium ion is adsorbed sufficiently to make the albumin positively ~ h a r g e d the , ~ behavior of the anions becomes normal. As a matter of fact Pauli observed that the solutions became slightly acid, indicating the adsorption of lime. We have still to consider the reversibility of the precipitation. In neutral or slightly alkaline solution the precipiJour. chim. phys., 5, 495 (1907). Pfluger’s Archiv.. 48, 132 (1891); cf. Hardy: Proc. Roy. Soc., 66,
IIO
(1900).
Hofmeister’s Beitrage zur chem. Physiol., 5, 2 7 (1901). Billitzer states t h a t the albumin does move to the cathode, Zeit. phys.
Chem., 51, I j j
(1905).
11'ildcv D . Baizcrqtt
356
tation by the salts of the alkalies is reversible; the precipitation by the salts of the alkaline earths is reversible if the precipitate is washed a t once with water but is irreversible if the precipitate is allowed to stand for a short time in contact with the solution; the precipitation by dilute solutions of salts of the heavy metals is irreversible. I n acidified solutions the precipitation by sulphocyanates and iodides is irreversible, while the precipitation by actetates and sulphates is reversible if the acid concentration is verj- low; for concentrations of o 03 S HC1 the precipitation is irreversible.' The precipitation of a colloid is always reversible in case no coalescence or agglomeration takes place, because one of the standard methods of preparing a colloidal solution is to wash out the excess of the precipitating agent. X o w the more strongly the precipitating agent is adsorbed, the harder it is to wash it out and consequent1)- the more nearly irrel-ersible the precipitation is. IVe could use this qualitatiT-ely t o account for the ,difference between precipitation by potassium sulphate and zinc sulphate, but we know that there must be agglomeration or coalescence when albumin is precipitated by lime salts because the precipitate can be peptonized when first formed and cannot be later. E\-en if we got round this, as we undoubtedly could, it is difficult to see how t o apply this explanation satisfactorily to the case of precipitation in acidified solution. It seems more probable that the physical properties of the precipitates vary under different conditions of precipitation and that the irreversible precipitates are more gelatinous than the others. TI-e know that the so-called Pkan de St. Gilles ferric oxide is precipitated by hydrochloric acid or nitric acid in a sandy form which is readily peptonized by water and is precipitated by sulphuric acid in a more gelatinous form which is not readily peptonized by mater. There are no data bearing directly on this point; but Pauli3 ~~
1
Pauli
Pfluger's Airchi\ , 5 ,
2j
( 1 9 0 4 , , Freundlich
Kapillarchemie, 425
(1909)
Bancroft Jour Phys Chem , 19,236 (191j). Pauli and Handoiiskp Biochern Zeit, 18, 340 (1909); Pauli and Falek Ibid , 47, 269 (1912) 2
3
Coagzilatioiz o j Albuiiiiiz bj' Electrolj'tes
357
has suggested in several papers that salts may change the hydration of suspended albumin. The only other point to be considered is the behavior of so-called electrolyte-free serum albumin. This shows no perceptible diffusion under electrical stress and is therefore apparently electrically neutral without being instable. Addition of sodium, barium, or calcium chloride does not make the albumin electrically positive or electrically negative. It is not precipitated by salts of zinc, copper, mercury, iron N a S C S causes no precipitaor lead. A mixture of CaC12 tion, though precipitation takes place if the albumin solution is acidified before the mixture is added. The only way that I see to account for the stability of the electrolyte-free albumin is to postulate that in the absence of salts the peptonizing action of water is enough to keep the albumin in colloidal solution.' To this extent the electrolyte-free albumin behaves like gelatine. This is not impossjble, because Denaeyer3 claims that when albumin is heated in water under a pressure of one atmosphere it becomes soluble again and reacts uncoagulated albumin. Granted that the electrolyte-free albumin is stable and electrically neutral, as Pauli claims, there is no reason why it should be precipitated bj- salts of zinc, copper, lead, mercury, iron, or lead because these salts would give it a positive charge and make it even more stable. I do no know why the chlorides of sodium, barium or calcium do not give it an electrical charge of one sign or the other, nor why the mixture of calcium chloride and sodium sulphocyanate does not precipitate it. The obvious assumption to make is that in the course of purification the albumin had been so changed by hydration, dehydration or otherwise, that it did not adsorb sodium, calcium, barium, chloride, or sulphocyanate ions to an appreciable extent; but does so after contact with an acid. Colloidal silver bromide has ap-
+
Pauli: Hofmeister's Beitrage zur chem. Physiol., 7, 531 (1906). I t is p o d o l e , however, t h a t the electrolyte-free albumin is the external phase in the solution. If so, this would account for some of its peculiarities. Jour Chem. SOC.. 60, 1269 (1891). 2
Wilder D . Bancrqft
358
parently practically no adsorbing power for potassium ions or nitrate ions whereas it does adsorb silver ions or bromide ions. All this is purely speculative and not worth much until there is some experimental evidence forthcoming. It does seem to me, however, that it will be an easier task t o account for the properties of electrolyte-free albumin on the basis of varying adsorption than on the basis of an amphoteric electrolyte. It is usually considered that albumin constitutes a special case1 and that it differs fundamentally from colloidal gold, let us say, or colloidal ferric oxide. We are told that positively charged colloids are precipitated by anions and that the nature of the cations is immaterial; we are also told that the cation is the all-important factor in the case of a negatively charged colloid, the nature of the anion being immaterial. This seems to be hopelessly wrong. M y whole argument will apply to any colloidal solution where the suspended phase becomes instable when electrically neutral ; and consequently the nature both of cation and of anion is important. The error has come about very naturally because it was very difficult to prepare a positive gold sol for instance or a negative ferric oxide sol. With a positively charged sol the cations which are less readily adsorbed than the ion causing the charge-usually hydrogen-naturally have very little effect. With a negatively charged sol the anions, which are less readily adsorbed than the ion causing the charge-usually hydroxyl, except perhaps in the case of the metals-naturally have very little effect.? Once the erroneous belief had taken root, it grew, because other people either did not test large numbers of cations and anions or ignored the evidence in case they did. I shall discuss this matter more in detail in another paper. The general conclusions of this paper are as follows: I . So far as the data go, the coagulation of albumin by salts can be accounted for more satisfactorily on the assumption that we are dealing primarily with adsorption rather 2
Freundlich : Kapillarchernie, 434 (1909). In extreme cases the adsorption may be so slight as to be negligible.
Coagulation of Albumin by Electrolytes
359
than on the assumption that we are dealing primarily with an amphoteric electrolyte. 2 . Slightly acid or slightly alkaline solutions of albumin are least stable when the dispersed phase is electrically neutral. 3. Since the sign of the electrical charge depends on the preferential adsorption of cation or anion, the nature of both anions and cations is important. 4. When albumin is charged positively, the more strongly adsorbed anion will be most effective in causing precipitation and the most strongly adsorbed cation in preventing precipitation. 5. When albumin is charged negatively, the most strongly adsorbed cation will be most effective in causing precipitation and the most strongly adsorbed anion in preventing precipitation. 6. If one adds to negatively charged albumin a salt consisting of a readily adsorbed cation and a slightly adsorbed anion, we shall get precipitation at low concentrations and no precipitation at higher concentration; but in this latter case the albumin will be charged positively. 7. While the reversibility of precipitation depends in part on the ease with which the precipitating agent can be washed out, it seems probable in the case of albumin that the physical properties of the precipitate are important as determining coalescence and agglomeration. There are no data on this point. 8. In the case of the electrolyte-free albumin one must apparently assume that the stability of the solution is due to the peptonizating action of water in the absence of electrolvtes. 9. The coagulation of albumin by electrolytes is a typical and general case, whereas the coagulations of gold sols and ferric oxide sols are special cases. Coriiell CiziLcvsiiy
