T H E INFLUENCE OF GELATIN AND ELECTROLYTE CONCENTRATION ON T H E RATIO OF ELECTRO&3MOTIC TO ELECTROPHORETIC MOBILITY1 H. L. WHITE AND LYMAN FOURT Departments of Physiology and of Zoology, Washington University, St. Louis, Missouri
Received August 1 , 1997
While it has been repeatedly shown that in salt solutions of concentration greater than 1 x 10-2 molar the ratio of electroosmotic to electrophoretic velocity at a gelatin-aqueous interface is unity, White, Monaghan, and Urban (8) presented evidence that this holds true only for electrolyte concentrations above the range 10-8 to molar. At concentrations below this range the ratio of the velocities departs from unity, becoming as high as 2.0. Bull (2) and Moyer and Abramson (7), however, state that with protein surfaces the ratio is unity, even in very dilute salt solutions (conductivities corresponding to 1 to 4 X M potassium chloride). The present communication is a partial repetition and an extension of the experiments of White, Monaghan, and Urban and of Moyer and Abramson to investigate the reason for the apparent discrepancy. The present report presents evidence that the observations both of White, Monaghan, and Urban and of Moyer and Abramson are correct, but are not comparable, owing to the influence of the concentration of gelatin employed. MET H0D6
A cylindrical Pyrex Mattson-type cell was used. The gelatin was adsorbed on powdered Pyrex particles 1 to 3 micra in diameter. The cell was always allowed to stand at least a half hour, usually an hour or more, filled with the experimental solution, or with this slowly passing through it. The preparations of gelatin used were as follows: Eastman Purified, ash content 0.03 per cent; Coignet “Silver Lable” ; Afga “Lichtfilter” (Ucopoco 6415 “with low ash content”), ash 1.10 per cent. The filtered 1 or 2 per cent stock solutions were kept in the ice box when not in use, and were used within two days or so of preparation. Observations of the time to traverse a given distance were made and recorded by the same observer. The average was taken of five traverse times in each direction. The mean velocity for a given depth was call
Presented at the Fourteenth Colloid Symposium, held at Minneapolis, Minne-
sota, June 10-12, 1937.
29
30
H. L. WHITE AND LYMAN FOURT
culated from the mean velocities in the two directions. Drift was usually small; in one experiment for which the deviations of the times in each direction were calculated, the deviation was 4.2 per cent. Readings were taken at a number of levels in the cell, as recommended by Moyer and Abramson. It can be shown that the equation (1)
Y = ~ ( Z - X ' ) + C
used by them for the flat cell describes the observed velocity at any level in the round cell as well2;y is the observed velocity at any fraction, z,of the diameter from the top; b is the slope of the resulting straight line, and c is the intercept at z = 0, Le., the velocity at the wall. For any given solution, b and c should be constants. Since at the wall yo = c, c is the difference between the electroosmotic and electrophoretic velocities. In the round cell the ratio is
R=u -= V
y0.6
- yO.147 = y0.14T
_ I _ _
- YO
y0.147
y0.147
(2)
where the subscripts refer to the fraction of the diameter from the top at which the velocity was observed, and u is the electroosmotic, v the electrophoretic velocity. The values of c, R, u,and v, given in tables 1 and 2 arise not from any single pair of observations but from an application of equation 1 to points obtained throughout the upper half of the cell. In y0.147, or yo used to determine R according to every case the values of equation 2 are obtained graphically from the straight-line plots of equation 1, as is illustrated in figures 2 and 3. The values of y0.147 and YO4 80 obtained agree closely with the values directly observed at these levels. The conclusion that R was unity where there was no observable movement near the wall was confirmed graphically. 2
Mattson (J. Phys. Chem. 37,223 (1933)) gives the equation
for the velocity, V , of the liquid a t any radius, r , from the center in a tube of radius a, c heing a constant. The fraction of the diameter measured downwards from the top is
The observed velocity, y, of a particle is the sum of v, its electrophoretic velocity, and V . In terms of z we have y = V
+ v = -4caP
(z
- 9)+ +caz + v
which is of the same form as equation 1 of the text, in which b is -4caz and c i s tea* v.
+
RATIO OF ELECTRO~SMOTIC TO ELECTROPHORETIC MOBILITY
31
EXPERIMENTAL
Tests of completeness of coating of walls and particles For the present argument it is essential to know that a gelatin concentration of 0.01 per cent produces an effective coating on both glass particles and cell wall. While it has been the universal finding that this concentration is sufficient to ensure adequate coating (1, 4, 6), two types of experiments were carried out to test this point. First, if the cell is allowed to stand filled with 1 per cent gelatin in distilled water for an hour, then rinsed with 0.01 per cent geIatin in distilled water, the ratio is still well above unity; in one case of three the ratio was not altered by previous cell treatment with 1 per cent gelatin, in two cases it was somewhat lowered, presumably because of adventitious factors. Also, in the case of the experiments with 0.02 per cent Eastman gelatin given in table 2 the cell had stood for an hour before the experiment filled with 1 per cent gelatin, yet the ratio is clearly greater than unity. We confirm earlier findings that equilibrium conditions are established within an hour. Second, simultaneous determinations of electrophoretic and electroosmotic isoelectric points were carried out. If the cell is incompletely coated while the particles are completely coated there will be electroosmotic movement with no electrophoretic movement. A series of 0.01 per cent solutions of Eastman’s gelatin was prepared from the same stock solution. All were 2 X 10-4 M in added chloride, with various proportions of hydrochloric acid and potassium chloride to vary the pH, which was determined with the glass electrode. The results are shown in figure 1. Plotting u, electroosmotic velocity, and v, electrophoretic velocity, against pH gives smooth curves, both of which cross the axis of zero mobility a t p H 4.75. The objection might be raised that even though a concentration of 0.01 per cent gelatin coats the cell wall sufficiently to make the wall behave as a gelatin surface when a t its isoelectric point, the coating may still not be adequate to make the wall behave as a gelatin surface when seta is other than zero, while the broken particle surfaces would be adequately coated. That is, the advocates of the view that the ratio is always unity, given identical surfaces, might say that our findings are due merely to incompleteness of the cell wall coating as compared with the particle coating. 4 further reason, however, for believing that the cell wall is in fact completely coated in 0.01 per cent gelatin is the finding of Dummett and Bowden and of ourselves that electroosmotic velocity a t constant p H falls as gelatin concentration is increased from zero and becomes constant a t a concentration between 0.001 and 0.01 per cent, Le., the electroosmotic velocity changes from that of bare glass to that of gelatin, the latter being attained a t between 0.001 and 0.01 per cent gelatin. The argument must
32
H. L. WHITE AKD LYMAK FOURT
be confined to completeness of coating of the cell wall; it cannot be maintained that the particles are less completely coated than the cell wall, for in that case electrophoretic velocity would be greater than electroosmotic. Monaghan and White (5) found that when one uses microscopic glass particles with fused surfaces the ratio of elwtroosmotic to electrophoretic velocity is greater than unity in potassium chloride solutions of 10-3 M or lower concentration. Here we have a case where wall and particle surfaces are known to bP identical; the observation has a rather direct bearing on the point under discussion, in that it shows that 0
UECTWPMXESIS EcECTR005UCSS
.;i
s
0
ci
c
c
01
DH
5
I
1
d/
'f
NJL
FIG.I
FIG.2 FIG.1. pH-mobility curves of electrophoresis and electroosmosis in 0.01 per cent Eastman gelatin, showing identity of isoelectric points. FIG.2. Effect of varying gelatin (Eastman) concentration in distilled water on mobilities and ratio of mobilities. The function (z- 9)of equation 1 is plotted on the horizontal axis. The y intercept shows the excess of electroosmotic over electrophoretic mobility. The arrow indicates the level, z = 0.147, of true electrophoretic mobility. The mobility a t the center of the cell, where z - x* = 0.25, is the sum of electroosmotic and electrophoretic mobilities. The numbers of the various curves refer t o the experiment numbers of table 1.
it is possible to have a greater than unity ratio with identical surfaces, although it does not directly prove the case for gelatin. The matter seems worth discussing, aside from its purely theoretical implications, because of its rBle in the explanation advanced by tlihite and Monaghan (9) of the common observa,tion that the critical potential (as determined by electrophoresis) seen with electrolytes which coagulate on low concentration is lower than that with those which require higher concentrations; the lower apparent critical potential with the polyvalent electrolytes is considered to be an artifact due to the impossibility of calculating the true zeta potential from electrophoretic mobility in dilute solutions.
33
RATIO OF ELECTROOSMOTIC TO ELECTROPHORETIC MOBILITY
Effect of gelatin concentration We find that varying the gelatin concentration, in distilled water solution, has a very marked effect (table 1 and figure 2). In every case, concentrations of 0.08 per cent oremore give low mobilities, and ratios of unity. For concentrations of 0.01 to 0.02 per cent gelatin, except Agfa gelatin, the electroosmotic mobility is the larger, the ratio ranging from 1.13to 1.78. The spread of values a t a given concentration (0.01per cent) in different experiments must be due t o the combination of a number of TABLE 1 Effect of gelatin concentration i n distilled water EXPP. NO.
QELATTIN
R
PH
6.32
mima per second per volt per cm.
am8 per 100 cc
1 2 3 4 5
Eastman 0.01 0.01 0.02 0.09 0.2
1.53 0.78 0.32 0 0
3.57 2.88 1.73 0.68 0.135
2.04 2.10 1.41 0.68 0.135
1.75 1.37 1.23 1.oo 1.oo
6 7 8 9 10 11 12
Coignet 0.01 0.01 0.01 0.01 0.02 0.1 0.2
1.82 1.05 1.73 0.70 0.66 0 0.02
4.29 3.52 3.80 1.98 2.32 0.64 0.475
2.43 2.48 2.20 1.28 1.64 0.64 0.45
1.76 1.42 1.77 1.55 1.41 1.oo 1.06
13 14 15 16 17
Agf a 0.01 0.02 0.05 0.1 0.2
0 0 0 0 0
0.45 0.45 0.20 0.20 0.07 0.07 0.06 0 .OB Very low
1.oo 1.oo 1.oo 1.oo 1.oo
4.87
6.12 5.59 5.28
6.80
6.21
uncontrolled factors, such as the following: protein concentration itself, within small limits; accidental electrolyte contamination; age and history of the gelatin solution; temperature.
E$ect
of
electrolytes
Table 2 shows that the ratio of the two mobilities is slightly more than unity in lo-* M potassium chloride, but is unity in 10P M , confirming the results of White, Monaghan, and Urban (8). Both Coignet and Eastman gelatins, the latter in two concentrations, 0.01 and 0.02 per cent, show this
34
wnIm
H. L.
AND LYMAN FOURT
effect. Comparison of the two gelatin concentrations in each concentration of added electrolyte shows a lower velocity for higher gelatin conTABLE 2 Bflect of added eleelrolvte . I _ _ _
ADDED
1A 1B 1c 1D 3A
Eastman 0 01 0 01 0 01 0 01 0 02
KCI
PH
molar
micra ppr second per zolt per cm.
0 10-4 10-3 10-2
1.60 0.93 0.20 0 0.32 0.40 0.17 f
3.65 2.25 0.99 0.49 1.73 1.40 0.77 0.32
1.05 0.90 -0.43 0 0.70 0.34 0.09
3.52 3.18 1.43 0.72 1.98 1.40 0.74
0
10-4 10-3 10-2
0
10-4 10-3 10-2
0
2
x
10-4
10-3
0.49 1.41 1.00 0.60 0.36 2.48 2.28 1.85 0.72 1.28 1.06 0.64 ~
1
1.00 1.23 1.40 1.28 0.92
5.15 5.00 4.86
1.42 1.39 0.77 1.00 1.55 1.32 1.16 _
" rI KCL 0
8
0
0
I
x I0-W
e
I X W ~ M
0
1 X IV2M
5
N L
FIG.3. Effect of varying concentration of added electrolyte on mobilities and ratio of mobilities of 0.01 per cent Eastman gelatin. The numbers of the curves refer t o the experiment numbers of table2.
centration, as did the experiments on gelatin in distilled water. Figure 3 shows straight-line plots of experiments with 0.01 per cent Eastman gelatin.
_
RATIO OB ELECTROOSMOTIC
TO ELECTROPHORETIC MOBILITY
35
Digerences in the gelatins Since we did not dialyze any of these preparations, some differences due to the ash content may be expected. The specific conductances of 0.1 p’er cent solutions of these gelatins were measured in a separate set of experiments, and were found to be 7.67,30.6, and 36.2 X 10-6mhos for Eastman, Coignet, and Agfa gelatin, respectively. Neither the makers’ stated ash content nor the determined conductivity suffices to account for the low mobility of our sample of Agfa gelatin; it must be attributed to the intrinsic properties of this particular protein. DISCUSSION
Four of the six experiments on gelatin reported by Moyer and Abramson were done with gelatin concentration of 0.2 per cent, one with 0.05 per cent gelatin, and one with 0.02 per cent gelatin, the last showing the highest value of the ratio found by them, 1.11. Bull’s experiments were done with gelatin concentrations ranging from 0.158 to 0.219 per cent, and were all M hydrochloric acid. Since White, Monaghan, and in 3 to 8 X Urban used a gelatin concentration of 0.01 per cent (Eastman purified gelatin, no dialysis) the present series of experiments shows that there is no incompatibility in the results of the various w o r k e r ~ . ~Moyer and Abramson’s velocities are expressed only in relative units, and so do not permit a quantitative comparison. That the reversal near the wall is not due to any specific wall effects, as suggested by Moyer and Abramson to account for their occasional observation of this same phenomenon, is seen from the fact that the value of c a t the wall is determined not solely by points taken near the wall, but is consistent with mobilities a t depths all the way down to the center. The nearest approach to the wall which it is feasible to make is a matter of 0.01 to 0.02 mm., the cell diameter being 2.420 mm. The present experiments confirm the previous work of White, Monaghan, Since this paper was written NIoyer, working in Abramson’s laboratory, informs us that he finds that with 0.01 per cent and even with 0.001 per cent gelatin the ratio is usually unity, provided that the particles are first treated with a stronger gelatin solution, and sometimes even without such pretreatment His interpretation is that when adequate measures are taken t o insure complete coating of both surfaces the ratio is unity. We find, on the other hand, the ratio greater than unity with 0.01 per cent gelatin even though both cell and powder have first been treated with 1 per cent gelatin and then allowed to come to equilibrium with 0.01 per cent. It seems to us most probable that differences in the nature or previous treatment of the gelatins must be responsible for the discrepancies of observation; it will be noted that our Agfa gelatin always showed unity ratio even with 0.01 per cent concentration in distilled water. The fundamental question of whether a ratio greater than unity, when it is observed, is due to a real difference in the electrokinetics of electrophoresis and of electroosmosis, respectively, or merely to a lack of identity of surfaces is still not answered t o the satisfaction of all concerned. THE JOURNAL OF PHYSICAL CHEMIBTRY, VOL. 42, NO. 1
36
H. L. WHITE AiYD LYMAN FOURT
and Urban in showing the electrophoretic velocity to be lower than the electroosmotic in low gelatin and electrolyte concentrations; they also confirm the work of Bull and of Moyer and Abramson, in finding the velocities the same with higher gelatin but unchanged electrolyte concentration. The essential difference between the two sets of experiments seems to lie in the concentration of gelatin. No satisfactory explanation of the reaSon for this effect of gelatin concentration is apparent; however, its bimilarity to that of added inorganic electrolyte suggests that the gelatin, by virtue of its ionization as zwitter ions, is itself the equivalent of added electrolyte. The zwitter-ion electrolyte, although often stated to have the properties of a strong electrolyte, would not be revealed by the conductivity measurements which have usually served as criteria for freedom from electrolytes. The suggestion may be given a rough quantitative test. According to the compilation of Czarnetsky and Schmidt TABLE 3 Effect of added m i l t e r ions 0.01 per cent Eastman gelatin in 1 X lo-* M acetate buffer GPBCIFIC C O I -
ADDED GLYCINE
U
________-_
AT
x 10-5 x 10-4 x 10-3 1 x 10-2 5 x 10-2 2 x 10-1
5 5 5
5 39 5 45 5 35 5 44 5 21 5.50
micra per second per volt per cm.
1.39 1.40
R
____
~
mhos X 105
molar
0
25°C.
_ _ _ - ~
2.19 '
1
1.91 1.25 1.65
' ~
1.05 1.65
1.74 1.68 1.40 1.15 1.19 1 .GO
1.15 -
(3), the total acid-binding capacity of gelatin is 89 x 10-5 moles per gram; the total base-binding rapacity is 64 X lop5 moles per gram, averaged from conductimetric and potentiometric determinations. These experimentally determined values agree roughly with those calculated by them from the known amino acid composition. On this basis, a t the isoelectric point there are present 64 X moles of zn-itter ions per gram of gelatin, and a 0.1 per cent solution is 6.4 X molar in zwitter-ions, a t a maximum. Since the concentration of potassium chloride required to bring the ratio of the two velocities to unity falls between 10-3 and 10-2 molar, it is evident that if the gelatin is acting as a zwitter-ion electrolyte, it is either about twice as effective as is potassium chloride, or it has an additional effect of some other nature. In order to test experimentally the concept that zwitter ions may affect electrophoretic mobility, the influence of added glycine in various concentrations on the ratio of electroosmotic to electrophoretic mobility of
RATIO O F ELECTROOSMOTIC TO ELECTROPHORETIC MOBILITY
37
0.01 per cent Eastman gelatin was determined. The values of 11 and v in this series were determined by observations a t the 50 and 14.7 per cent cell diameter levels. The findings are shown in table 3. I t is evident that added glycine, although it has no significant effect on conductivity of the solutions employed, acts as does added potassium chloride in lowering the ratio to unity. The supposition is thuq strengthened that the unity ratio seen in high gelatin concentrations, as 0.1 or 0.2 per cent, is due to the same mechanism. SUMMARY
Evidence is presented that 0.01 per cent gelatin is sufficient to coat completely the suspended particles and cell wall, giving characteristic protein surfaces; our former finding is confirmed that here the ratio is greater than unity in potassium chloride solutions of concentration M or lower. Evidence is presented that the ratio of electroosmotic to electrophoretic mobility is dependent on the concentration of gelatin as well as of added electrolyte. The effect of added gelatin is qualitatively the same as that of added electrolyte and is apparently due to its zwitter-ion concentration. REFERENCES (1) ABRAMGOS, H. A : J. Am. Chem. SOC.60,390, 3389 (1928). (2) BULL,H. B.: J. Phys. Chem. 39,577 (1935). C. L. A. : J. Biol. Chem. 106, 301 (1934). (3) CZARNETZKY, E . J., AND SCHMIDT, (4) DUMMETT, A . , ANI) BOWDEN, P.: Proc. Roy. SOC.(London) A142,382 (1933). (5) R ~ O N A G H A N ,B.,-ANDWHITE,H. L. : J. Phys. Chem 39,935 (1935). (6) R ~ O N A G H A N ,B . , WHITE,H. L., AND URBAN,F. : J Phys Chem. 39,585 (1935). (7) NOYER, L. S., AND ABRAMSON, H. A. : J. Gen. Physiol. 19,727 (1936). (8) WHITE,H. I , . , ~ I O N A G H B., A NAND , URBAN,I?.: J. Phys Chem. 39,611 (1935). (9) WHITE,H. L., AND MOKAGHAN, B.: J . Phys. Chem. 39,925 (1935).
