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Physical chemical characterization of percoll. III. Sodium binding
Physical Chemical Characterization of Percoll III. Sodium Binding TORVARD C. LAURENT AND HfkKAN PERTOFT Institute o f Medical and Physiological Chemistry, University o f Uppsala, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden Received May 30, 1979; accepted October 10, 1979 The sodium binding to Percoll and Ludox HS has been measured by equilibrium dialysis of 22NaC1. The regular silica colloid, Ludox HS, bound two to three times more sodium ions than the polyvinylpyrrolidone-coated silica colloid, Percoll, at pH 7 and 8 and at sodium chloride concentrations of 0.2, 1, and 8.5 mM, respectively. Both colloids showed an increasing sodium binding with increasing pH and increasing ionic strength. The data did not differentiate between sodium bound to the colloid surface and the counterions to negative charges on the colloid. It was concluded that the polymer coat of Percoll diminished dissociation of silanol groups on the surface of the colloid. INTRODUCTION
The preceding papers describe the particle weight, size, and interaction of the colloid in Percoll (1, 2). The Percoll particles consist of a nucleus of silica and a shell of polyvinylpyrrolidone (PVP). It is well known that silica particles without a polymer coat carry a negative surface charge (3) and it is probable that the adsorbed organic compounds on its surface will interact with dissociable groups (4). For example, it has been shown that the addition of a neutral polymer to colloidal silica influences the pH of the solution (5). Furthermore, the electrophoretic mobility of colloidal silica is 10fold higher than that of Percoll over a large pH range (6). We have therefore compared the sodium-binding capacity of Percoll and its parent silica colloid, Ludox HS. The pure-silica colloid binds two to three times more sodium ions than Percoll at the pH values and ionic strengths studied. MATERIALS AND METHODS
Materials. Percoll (Batch 96544D) and a pure silica colloid, Ludox HS, were the same
as described in a preceding paper (1). Carrier-flee mNaC1 was from the Radiochemical Center, Amersham, England. Analytical techniques. Colloid concentrations were determined by dry weight measurements (1). Sodium was determined in a Perkin-Elmer 460 atomic absorption spectrophotometer. The analyses were kindly performed by Dr. H. Ulfendahl, Department of Physiology, University of Uppsala. Radioactivity was counted in a Packard Auto-Gamma scintillation spectrometer, Model 3001. Equilibrium dialysis. Sodium binding was studied by equilibrium dialysis. Stock solutions of PercoU and Ludox HS were prepared by adjusting two portions of each colloid to pH 7 and 8, respectively, with hydrochloric acid. The four solutions were then dialyzed separately against several changes of distilled water for 48 hr and their concentrations were determined. Each stock solution was used to prepare four series of samples. Each series contained a different concentration of sodium chloride (0 M, 1 raM, 8.5 raM, and 0.1 M) and within each series there were three different con-
142 0021-9797/80/070142-04502.00/0 Copyright© 1980by AcademicPress,Inc. All rightsof reproductionin any formreserved.
Journal of Colloidand InterfaceScience, Vol. 76, No. 1, July 1980
143
SODIUM BINDING TO PERCOLL
% x E
o. E m
e~
.o
I
0
.05
.10
.1.5
CONCENTRATION OF COLLOID ( g / m l )
FIG. 1. Demonstration of an equilibrium dialysis experiment. Percoll and Ludox HS solutions of varying concentrations were dialyzed simultaneously against 0.01 M NaC1, pH 7.0, containing 2~Na+. The outside fluid contained 7.033 cpm/ml after equilibration. The ordinate gives the amount of radioactivity bound to the colloid per milliliter of sample. It can be calculated from the linear correlations that Ludox HS (A) bound 38,700 and Percoll (0) 12,400 cpm/g colloid, respectively.
centrations of colloid (between 0.03 and 0.12 g/ml). The equilibrium dialysis was performed in eight closed Erlenmeyer flasks, each containing 150 ml of solvent. Four of the flasks were used for experiments at pH 7 and the others for experiments at pH 8. The solvents used at each pH were distilled water, 1 mM, 8.5 mM, and 0.1 M sodium chloride. Each flask was used for the simultaneous dialysis of the three Percoll and three Ludox HS samples of corresponding pH and ionic strength. Each sample had a volume of 5 ml. To each flask was added 0.2 nmole of 22NaC1 in 200 txliter (approximately 800,000 cpm). The dialysis was continued for 6 days at room temperature under continuous stirring.
The volume of each sample was measured at the termination of the dialysis and the concentration of colloid corrected for volume changes, pH and radioactivity were measured in all samples. Radioactivity and sodium concentration were determined in the bathing fluids. The amount of bound radioactivity in each sample was calculated as the difference between its total radioactivity and the radioactivity of its solvent. To obtain the amount of solvent in a sample the volume of the colloid was subtracted from the total volume. The volume of the colloid was calculated from its weight and partial specific volume (2). The radioactivity per unit volume of solvent in the sample was assumed to be the same as in the bathing fluid. The amount of radioactivity bound to the colloid was converted to sodium binding with the aid of the specific activity of the sodium in the bathing fluid. RESULTS
Preliminary experiments showed that dialysis equilibrium at the present conditions was reached within 1 day. The equilibration time chosen should therefore be more than sufficient. The results of an equilibrium dialysis experiment are demonstrated in Fig. 1. The amount of radioactivity bound to the colloid was always linearly correlated to the concentration of the colloid. Ludox HS always bound two to three times more radioactivity than Percoll. The results obtained in 0.1 M NaC1 could not be used for reliable calculations. The fraction of bound sodium was in this case too small compared to the amount of free sodium in the solution. Concentrations of sodium in the surrounding fluids as determined by atomic absorption were 0.169 and 0.265 mM after dialysis against distilled water at pH 7 and 8, respectively. The sodium concentration of the 1 mM solutions was also slightly higher than expected. The colloids did obviously Journal of Colloid and Interface Science, V o l . 7 6 , N o . 1, J u l y 1980
144
LAURENT AND PERTOFT TABLE I Binding of Sodium to Percoll and Ludox HS Number of charges per particle from Donnan equilibrium
Sodium bound by colloidal particles /~mole/g colloid
Na ions/particle
Na ions/cm 2.10 -12
Concn of NaC1 (mM)
Percoll
Ludox
Percoll
Ludox
Percon
6.85-7.00
0.2 1.02 8.5
14 15 21
32 44 64
90 100 130
210 290 420
7.82-8.20
0.2 1.02 8.5
24 31 30
58 81 96
160 200 190
380 530 630
pH
release small amounts of sodium during the dialysis. There were slight variations in the final p H of the samples dialyzed at various ionic strengths. The p H range is given in Table I. The sodium binding determined in the experiments is displayed in Table I. It has been e x p r e s s e d both in terms of micromoles of sodium bound per g r a m of colloid and n u m b e r of sodium ions bound per colloidal particle assuming a molecular weight for both Percoll and L u d o x H S of 6.5 × 10 8 (1). DISCUSSION The results in Table I tell us that the sodium binding per gram Percoll is two to three times lower than that of L u d o x HS. T h e y also show that the sodium binding a p p r o x i m a t e l y doubles for both colloids when the p H rises from 7 to 8. T h e r e is also an increase in sodium binding with increasing free sodium ion concentration but this is m o r e p r o n o u n c e d for L u d o x H S than for Percoll, The P V P coat on the Percoll surface does apparently prevent groups on the silica surface f r o m dissociation. This could be due to a decrease in the dielectric constant caused by the p o l y m e r in the surface layer. The sodium binding, as defined by the present technique, includes sodium ions bound to the surface structure, free sodium Journal of Colloid and Interface Science, Vol. 76, No. 1, July 1980
Ludox
Percoll
Ludox
6.8 7.6 9.9
18 25 36
100 150 190
230 370 520
12 15 14
33 45 54
180 260 270
410 610 730
ions which are counterions to negative charges, and the deviation of the sodium distribution caused b y the Donnan equilibrium. We can estimate if the Donnan equilibrium can be responsible for the data assuming all the sodium ions to be dissociated. If the activity coefficient of the salt is the same in both c o m p a r t m e n t s the Donnan equilibrium can be e x p r e s s e d b y the equation (see, e.g., Ref. (7)). (m~) 2 = m + ( m + - Z i n c ) , where m~ and m+ are the molal sodium ion concentrations in the solvent and solution c o m p a r t m e n t s , respectively, me the molal concentration of the colloid, and Z the number of negative charges on each colloidal particle. Calculation of Z was made f r o m the experimental values at a colloid concentration of 0.1 g/ml. m~ and m+ were calculated from the radioactivities in the solvent and solute c o m p a r t m e n t s , care being taken o f the volume correction for the colloid. The molecular weight 6.5 × 106 (1) was used to obtain the molal concentration of the colloid. The results are tabulated in Table I. It is a p p a r e n t f r o m the calculation that the n u m b e r of negative charges on the colloid particles, which one expects at an ideal Donnan equilibrium, only slightly exceeds the m e a s u r e d n u m b e r of sodium ions bound
145
SODIUM BINDING TO PERCOLL
per colloid particle. The experimental data can thus as well be explained by a complete dissociation of all sodium ions from the colloid surface and an ideal Donnan equilibrium as a direct binding of all the sodium ions to the colloid. Our data on Ludox HS can, however, be compared with the charge density of regular silica sols published by Bolt (8). At corresponding pH and sodium chloride concentrations our sodium binding is approximately twice as large as the number of charges. This would indicate that half of the sodium ions are directly adsorbed to the surface in Ludox HS. The results and knowledge of the particle sizes do also allow an estimate of the sodium binding (adsorbed ions + free counterions) per unit area. The weight-average diameters from electron microscopy of Percoll and Ludox HS are 20.5 and 19.3 nm, respectively (2). The sodium binding per unit area is tabulated in Table I. The values for Ludox HS are three to four times higher than those of Percoll. Although the electrophoretic experiments described earlier (6) are not directly comparable with the present experiments (the former were made at a higher ionic strength) it is interesting to note that Percoll had a mobility of only 10-15% of that of Ludox HS in the pH range 7 to 8, while the sodium binding of Percoll is 25-35% of that of Ludox HS. Similarly the mobility of both
Percoll and Ludox HS decreased only about 20% between pH 8 and pH 7 while the sodium binding decreased 40%. This is further an indication that there is a considerable amount of immobilized sodium especially in Percoll. Dissociated sodium ions could be present within the PVP coat of the Percoll particles and be part of the hydrodynamic unit moving in electrophoresis and thus act as if they were adsorbed to the surface. ACKNOWLEDGMENTS This project has been supported by the Swedish Medical Research Council through Grant 13X-4 and by Pharmacia Fine Chemicals AB. We are grateful to Dr. L. Khgedal for valuable discussions. REFERENCES 1. Laurent, T. C., Pertoft, H., and Nordli, O., J. Colloid Interface Sci. 76, 124 (1980). 2. Laurent, T. C., Ogston, A. G., Pertoft, H., and Cadsson, B., J. Colloid Interface Sci. 76, 133 (1980), 3. Iler, R. K., in "Surface and Colloid Science" (E. Matijevi6, Ed.), Vol. 6, p. 1. Wiley, New York, 1973. 4. Iler, R. K., in "Biochemistry of Silicon and Related Problems" (G. Bendz and I. Lindqvist, Eds.), p. 53. Plenum, New York, 1977. 5. Pertoft, H., Exp. Cell Res. 57, 338 (1969). 6. Pertoft, H., Laurent, T. C., L~tg~s,T., and K~tgedal, L., Anal. Biochem. 88, 271 (1978). 7. Tanford, C., "Physical Chemistry of Macromolecules," p. 225. Wiley, New York, 1961. 8. Bolt, G. H . , J . Phys. Chem. 61, 1166 (1957).
Journal of Colloid and Interface Science, Vol. 76, No. 1, July 1980
The preceding papers describe the particle weight, size, and interaction of the colloid in Percoll (1, 2). The Percoll particles consist of a nucleus of silica and a shell of polyvinylpyrrolidone (PVP). It is well known that silica particles without a polymer coat carry a negative surface charge (3) and it is probable that the adsorbed organic compounds on its surface will interact with dissociable groups (4). For example, it has been shown that the addition of a neutral polymer to colloidal silica influences the pH of the solution (5). Furthermore, the electrophoretic mobility of colloidal silica is 10fold higher than that of Percoll over a large pH range (6). We have therefore compared the sodium-binding capacity of Percoll and its parent silica colloid, Ludox HS. The pure-silica colloid binds two to three times more sodium ions than Percoll at the pH values and ionic strengths studied. MATERIALS AND METHODS
Materials. Percoll (Batch 96544D) and a pure silica colloid, Ludox HS, were the same
as described in a preceding paper (1). Carrier-flee mNaC1 was from the Radiochemical Center, Amersham, England. Analytical techniques. Colloid concentrations were determined by dry weight measurements (1). Sodium was determined in a Perkin-Elmer 460 atomic absorption spectrophotometer. The analyses were kindly performed by Dr. H. Ulfendahl, Department of Physiology, University of Uppsala. Radioactivity was counted in a Packard Auto-Gamma scintillation spectrometer, Model 3001. Equilibrium dialysis. Sodium binding was studied by equilibrium dialysis. Stock solutions of PercoU and Ludox HS were prepared by adjusting two portions of each colloid to pH 7 and 8, respectively, with hydrochloric acid. The four solutions were then dialyzed separately against several changes of distilled water for 48 hr and their concentrations were determined. Each stock solution was used to prepare four series of samples. Each series contained a different concentration of sodium chloride (0 M, 1 raM, 8.5 raM, and 0.1 M) and within each series there were three different con-
142 0021-9797/80/070142-04502.00/0 Copyright© 1980by AcademicPress,Inc. All rightsof reproductionin any formreserved.
Journal of Colloidand InterfaceScience, Vol. 76, No. 1, July 1980
143
SODIUM BINDING TO PERCOLL
% x E
o. E m
e~
.o
I
0
.05
.10
.1.5
CONCENTRATION OF COLLOID ( g / m l )
FIG. 1. Demonstration of an equilibrium dialysis experiment. Percoll and Ludox HS solutions of varying concentrations were dialyzed simultaneously against 0.01 M NaC1, pH 7.0, containing 2~Na+. The outside fluid contained 7.033 cpm/ml after equilibration. The ordinate gives the amount of radioactivity bound to the colloid per milliliter of sample. It can be calculated from the linear correlations that Ludox HS (A) bound 38,700 and Percoll (0) 12,400 cpm/g colloid, respectively.
centrations of colloid (between 0.03 and 0.12 g/ml). The equilibrium dialysis was performed in eight closed Erlenmeyer flasks, each containing 150 ml of solvent. Four of the flasks were used for experiments at pH 7 and the others for experiments at pH 8. The solvents used at each pH were distilled water, 1 mM, 8.5 mM, and 0.1 M sodium chloride. Each flask was used for the simultaneous dialysis of the three Percoll and three Ludox HS samples of corresponding pH and ionic strength. Each sample had a volume of 5 ml. To each flask was added 0.2 nmole of 22NaC1 in 200 txliter (approximately 800,000 cpm). The dialysis was continued for 6 days at room temperature under continuous stirring.
The volume of each sample was measured at the termination of the dialysis and the concentration of colloid corrected for volume changes, pH and radioactivity were measured in all samples. Radioactivity and sodium concentration were determined in the bathing fluids. The amount of bound radioactivity in each sample was calculated as the difference between its total radioactivity and the radioactivity of its solvent. To obtain the amount of solvent in a sample the volume of the colloid was subtracted from the total volume. The volume of the colloid was calculated from its weight and partial specific volume (2). The radioactivity per unit volume of solvent in the sample was assumed to be the same as in the bathing fluid. The amount of radioactivity bound to the colloid was converted to sodium binding with the aid of the specific activity of the sodium in the bathing fluid. RESULTS
Preliminary experiments showed that dialysis equilibrium at the present conditions was reached within 1 day. The equilibration time chosen should therefore be more than sufficient. The results of an equilibrium dialysis experiment are demonstrated in Fig. 1. The amount of radioactivity bound to the colloid was always linearly correlated to the concentration of the colloid. Ludox HS always bound two to three times more radioactivity than Percoll. The results obtained in 0.1 M NaC1 could not be used for reliable calculations. The fraction of bound sodium was in this case too small compared to the amount of free sodium in the solution. Concentrations of sodium in the surrounding fluids as determined by atomic absorption were 0.169 and 0.265 mM after dialysis against distilled water at pH 7 and 8, respectively. The sodium concentration of the 1 mM solutions was also slightly higher than expected. The colloids did obviously Journal of Colloid and Interface Science, V o l . 7 6 , N o . 1, J u l y 1980
144
LAURENT AND PERTOFT TABLE I Binding of Sodium to Percoll and Ludox HS Number of charges per particle from Donnan equilibrium
Sodium bound by colloidal particles /~mole/g colloid
Na ions/particle
Na ions/cm 2.10 -12
Concn of NaC1 (mM)
Percoll
Ludox
Percoll
Ludox
Percon
6.85-7.00
0.2 1.02 8.5
14 15 21
32 44 64
90 100 130
210 290 420
7.82-8.20
0.2 1.02 8.5
24 31 30
58 81 96
160 200 190
380 530 630
pH
release small amounts of sodium during the dialysis. There were slight variations in the final p H of the samples dialyzed at various ionic strengths. The p H range is given in Table I. The sodium binding determined in the experiments is displayed in Table I. It has been e x p r e s s e d both in terms of micromoles of sodium bound per g r a m of colloid and n u m b e r of sodium ions bound per colloidal particle assuming a molecular weight for both Percoll and L u d o x H S of 6.5 × 10 8 (1). DISCUSSION The results in Table I tell us that the sodium binding per gram Percoll is two to three times lower than that of L u d o x HS. T h e y also show that the sodium binding a p p r o x i m a t e l y doubles for both colloids when the p H rises from 7 to 8. T h e r e is also an increase in sodium binding with increasing free sodium ion concentration but this is m o r e p r o n o u n c e d for L u d o x H S than for Percoll, The P V P coat on the Percoll surface does apparently prevent groups on the silica surface f r o m dissociation. This could be due to a decrease in the dielectric constant caused by the p o l y m e r in the surface layer. The sodium binding, as defined by the present technique, includes sodium ions bound to the surface structure, free sodium Journal of Colloid and Interface Science, Vol. 76, No. 1, July 1980
Ludox
Percoll
Ludox
6.8 7.6 9.9
18 25 36
100 150 190
230 370 520
12 15 14
33 45 54
180 260 270
410 610 730
ions which are counterions to negative charges, and the deviation of the sodium distribution caused b y the Donnan equilibrium. We can estimate if the Donnan equilibrium can be responsible for the data assuming all the sodium ions to be dissociated. If the activity coefficient of the salt is the same in both c o m p a r t m e n t s the Donnan equilibrium can be e x p r e s s e d b y the equation (see, e.g., Ref. (7)). (m~) 2 = m + ( m + - Z i n c ) , where m~ and m+ are the molal sodium ion concentrations in the solvent and solution c o m p a r t m e n t s , respectively, me the molal concentration of the colloid, and Z the number of negative charges on each colloidal particle. Calculation of Z was made f r o m the experimental values at a colloid concentration of 0.1 g/ml. m~ and m+ were calculated from the radioactivities in the solvent and solute c o m p a r t m e n t s , care being taken o f the volume correction for the colloid. The molecular weight 6.5 × 106 (1) was used to obtain the molal concentration of the colloid. The results are tabulated in Table I. It is a p p a r e n t f r o m the calculation that the n u m b e r of negative charges on the colloid particles, which one expects at an ideal Donnan equilibrium, only slightly exceeds the m e a s u r e d n u m b e r of sodium ions bound
145
SODIUM BINDING TO PERCOLL
per colloid particle. The experimental data can thus as well be explained by a complete dissociation of all sodium ions from the colloid surface and an ideal Donnan equilibrium as a direct binding of all the sodium ions to the colloid. Our data on Ludox HS can, however, be compared with the charge density of regular silica sols published by Bolt (8). At corresponding pH and sodium chloride concentrations our sodium binding is approximately twice as large as the number of charges. This would indicate that half of the sodium ions are directly adsorbed to the surface in Ludox HS. The results and knowledge of the particle sizes do also allow an estimate of the sodium binding (adsorbed ions + free counterions) per unit area. The weight-average diameters from electron microscopy of Percoll and Ludox HS are 20.5 and 19.3 nm, respectively (2). The sodium binding per unit area is tabulated in Table I. The values for Ludox HS are three to four times higher than those of Percoll. Although the electrophoretic experiments described earlier (6) are not directly comparable with the present experiments (the former were made at a higher ionic strength) it is interesting to note that Percoll had a mobility of only 10-15% of that of Ludox HS in the pH range 7 to 8, while the sodium binding of Percoll is 25-35% of that of Ludox HS. Similarly the mobility of both
Percoll and Ludox HS decreased only about 20% between pH 8 and pH 7 while the sodium binding decreased 40%. This is further an indication that there is a considerable amount of immobilized sodium especially in Percoll. Dissociated sodium ions could be present within the PVP coat of the Percoll particles and be part of the hydrodynamic unit moving in electrophoresis and thus act as if they were adsorbed to the surface. ACKNOWLEDGMENTS This project has been supported by the Swedish Medical Research Council through Grant 13X-4 and by Pharmacia Fine Chemicals AB. We are grateful to Dr. L. Khgedal for valuable discussions. REFERENCES 1. Laurent, T. C., Pertoft, H., and Nordli, O., J. Colloid Interface Sci. 76, 124 (1980). 2. Laurent, T. C., Ogston, A. G., Pertoft, H., and Cadsson, B., J. Colloid Interface Sci. 76, 133 (1980), 3. Iler, R. K., in "Surface and Colloid Science" (E. Matijevi6, Ed.), Vol. 6, p. 1. Wiley, New York, 1973. 4. Iler, R. K., in "Biochemistry of Silicon and Related Problems" (G. Bendz and I. Lindqvist, Eds.), p. 53. Plenum, New York, 1977. 5. Pertoft, H., Exp. Cell Res. 57, 338 (1969). 6. Pertoft, H., Laurent, T. C., L~tg~s,T., and K~tgedal, L., Anal. Biochem. 88, 271 (1978). 7. Tanford, C., "Physical Chemistry of Macromolecules," p. 225. Wiley, New York, 1961. 8. Bolt, G. H . , J . Phys. Chem. 61, 1166 (1957).
Journal of Colloid and Interface Science, Vol. 76, No. 1, July 1980