- Email: [email protected]
A simple method for the characterization of the silica sols
A Simple Method for the Characterization of the Silica Sols Although inorganic colloid solutions are very important there are only a few physicochemical methods for their characterization, especially for the determination of the size and size distribution of the particles. In addition, these methods--such as electron microscopy, light scattering, or sedimentation--require sophisticated equipment and often the results may be questioned as to sample preparation or mathematical modelling used in the evaluation of the experimental data. Size exclusion chromatography (1), on the other hand, allows the investigation of the silica sols as they stand and the resulting elution profiles give direct evidence of the hydrodynamic radii (rn) of the particles as well as of their size distribution (2-4). However, the porosity of most of the gel exclusion media does not allow the analysis (separation) of the particles with rn bigger than 3-4 nm. Sepharose 4B gel has a resolution range up to several dozen nanometers but at elution volumes close to the void volume the resolution is rather poor. (This is due to the fact that in the standardization procedure (5), where one plots diameter versus the inverse probability integral, i.e., eft-l(1 - K) where the distribution coefficient K = (Ve Vo)/(Vt - Vo) with Ve, Vo, Vt being elution, void, and -
total volume, respectively, the value of (1 - K) is approaching the value of 1 where erf-l(1 - K) is rather independent of the argument.) By the use of a novel gel exclusion medium, Sephacryl S 1000, even bigger particles can be analyzed because between V0 (where the particles with 2rn >~ 300 nm elute) and Ft (with 2rn ~< 70 nm) all particles with sizes in this range are separated. Recently, this gel was successfully applied to vesicles where the diameter ranges between several dozen and several hundred nanometers (1). In this study we wanted to determine the size and size distribution of the silica sol particles by size exclusion chromatography using Sephacryl S 1000 and Sepharose 4 B CL gels. The columns were packed at a flow rate 4 ml/ h. After packing they were run with the elution solvent (0.01 MNaCI with NaOH added to pH = 9) for 2 days. For the columns 40 X 0.7 cm approximately ¼- ½ ml of 10 wt% silica sols (diluted immediately before the experiment) were chromatographed with a flow rate of 6 ml/h. The elution profiles were analyzed in fractions of ½- 1 ml by turbidimetry at 340 nm or by dry weight determination. Fast qualitative (but very sensitive) analysis was also made by the naked eye investigation of the Tyndall effect using
1
0
.
200
0
150
2O
15
/ J
IE
u.=
~
/// // o
100
O -
,.=, 10..~,
i/t3
.¢z c=l
c==
5O
'II
v
I
0.5
I
1
err-l(1 - g ]
1.0
1
I
{{
1.5
FIG. 1. Calibration curves of Sephacryl S 1000 (©) and Sepharose 4B CL ([3) columns. The equilibrium constant for the distribution of particles between the gel pores and fluid phase, K = (Ve - Vo)/(Ft - 1Io) w a s calculated from the positions of peaks of standards: for Sepharose column myoglobin (rH = 2 nm), aldolase (4.8 nm), ferritin (6.1 nm), and thyroglobulin (8.5 rim) were used while Sephacryl was calibrated with aldolase, ferritin, thyroglobulin, Ludox WP (rn = 1.05 nm) and latex beads (42.5, 50.5, and 110 nm coated with SDS).
282 0021-9797/86 $3.00 Copyright © 1986 by Academic Press, Inc. All fights of reproduction in any form reserved.
Journal of Colloid and Interface Science, Vol. 110, No. 1, March 1986
NOTES
:
V°
283
1
,
Vt
E c:
0.5
0.2 0.1 t,
B
B
10
12
14
lfi
EFFLUENT[g ]
FIG. 2. Elution profiles from Sephacryl S 1000 of different silica sols: Ludox TM (©), Tosil 1980 (Fq), and Tosil 1984 (A).
a laser beam (He-Ne, ~ = 632.8 nm). The Sephacryl S 1000 (Pharrnacia Fine Chemicals AG, Ztifich, Switzerland) column was calibrated with Dow Latex microspheres (Serva Feinbiochemica, Heidelberg, Federal Republic of Germany) with diameters 481 + 1.8, 364 + 2.4, 220 _+ 6.5, 109 _+ 2.7, and 85 _+ 5.5 nm with sodium dodecyl sulfate (SDS) added to eluent at a concentration of 0.1%. V0 was determined with 481-nm beads coated with SDS (the 364nm beads elute within experimental error at the same V=) while for Vt c ytochrome c, colored inorganic salts or complexes were used. Sepharose 4B CL (Pharmacia Fine Chemicals AB Uppsala, Sweden) was calibrated with 220and 85-rim beads (V0), cytochrome c and CuSO4 (Vt) and
with the proteins myoglobin, aldolase, ferritin and thyroglobulin, which were also used for the first column. The obtained standardization curves (5), 2rn(nm) = 37 erf-~(1 - K) for the Sepharose and 2rn(nm) = 155 err-l(1 - K) 35 for the Sephacryl are almost identical to the reported ones in the literature (6-8). Only in the region below erf-~(1 K) = 0.5 we approximated the standardization curve of Sephacryl column with the slightly concave exponential curve 2rn(nm) = 75 exp(3.6 eff-~(1 - K)). This curve is the best fit in the region where Ve ~ V0 and is similar to the reported one (7) except that it intercepts the ordinate closer to the real cut offdiameter. The calibration curves are shown in Fig. 1.
E (-,-)
OJ
.<
0.2
0.1
4
6
B
10
12
14 EFFLUENI[g]
FIG. 3. Elution profiles from Sepharose 4B CL of different silica sols: Tosil 1984 (A) and silica sol prepared without growth of particles (O).
Journal of Colloid and Interfac,e Science, Vol. 110, No. 1, March 1986
284
NOTES r=
l Vt
"~ 0.~ 2:
~ 0.3 N "~ Od
0.! ,
2
z,
G
8
10
12
14 EFFLUENT[g]
FIG. 4. Elution profiles from Sephacryl S 1000 of silica sols prepared with exaggerated (@) and without growth (©) of the particles and of aged Ludox HS (×).
Figure 2 shows some typical elution profiles of the standard silica sols on the Sephacryl S 1000 column. Average diameters of solutions of Ludox TM (O) (du Pont de Nemours, Wilmington, DEE), Tosil (n) (Chemapol, Prague, Czechoslovakia, produced in 1980), and Tosil (A) (1984) are 22.4, 21.3, and 14 nm, respectively. Because
these particles elute close to Vt they can be analyzed also by the Sepharose 4B, or Sepharose 2B (9), column and some typical elution profiles are shown in Fig. 3. The obtained diameters, 15.5 nm for Tosil 1984 (A) and 10 nm for the silica sol prepared in this laboratory (©) (see below) are in good agreement with the Sephacryl S 1000 results.
i,'
\\
///
I x\
~-.
0.20
"\
020
,
\ \
/
0.15 /
,~0.10
0.15
r
I-r--
..0=OlD
~
"~ 0.05
12
10
20
24
28
DIAMETER [ nm ]
32
6
10
14
18
22
DIAMETER [ nm ]
FIG. 5. Size distribution histograms (ndnt as a function of diameter, number of counted particles nt ~ 300) of (A) Tosil (1980) and (B) Tosil (1984) as determined by electron microscopy (samples were diluted to 0,1% and immediately deposited on carbon coated grids). The broken line represents the apparent size distribution of the chromatographic peaks. (Artificially, we applied the same procedure as was used for the peaks to all the points of the elution profile nevertheless the distribution coefficient K has physical meaning only at the equilibrium value, i.e., the top of the peak. This simplification also neglects the contribution of the diffusion and possible experimental parameters to the line broadening which is attributed solely to the superposition of the lines of heterogeneous sample (4). The resulting linewidth is at half-height of the ehition peak more than I5 nm and increases when V=approaches Vo.) These distribution curves indicate that in most cases the natural linewidth is too broad to allow the determination of the size distribution,
Journal of Colloid and Interface Science, Vol. 110, No. 1, March 1986
NOTES The elution profiles of some homemade silica sols on Sephacryl S 1000 column are shown in Fig. 4. The first one (O) was prepared without growth.of particles (to pH = 9 neutralized acidic silica sol was concentrated without heating at constant volume) while for the second one (O) the particles were grown (the diluted silica sol, pH = 9, was heated at constant volume for 2 h and concentrated to a very viscous sol which gelled overnight). The latter shows a very broad, heterogeneous profile with particles up to diameter of 175 nm while the former consists of very small particles (2rH = 9 nm, see also Fig. 3). The aged Ludox HS sample (×) also shows large particles (2rn = 166 nm). The results are in agreement with EM micrographs, In Fig. 5 the frequency distribution of the two Tosil samples is presented. The size of the particles determined by dectron microscopy and by size exclusion chromatography is in good agreement while the size distribution of the particles cannot be quantitatively determined because the natural linewidth of the elution peaks is more than ~ 15% of the column volume already for very homogeneous partides. Therefore, besides qualitative estimates from the widths of the peaks, these measurements can provide more evidence of the size distribution only for very heterogeneous samples (see also text of Fig. 5). The results indicate that size exclusion chromatography with commercially available gels is a simple and accurate method for the determination of the size of sol particles. Size distribution of sol particles can be determined only when the samples are very heterogeneous. However, due to the~slight changes in their properties during the usage, columns should be recalibrated after several runs if the quantitative analysis is desired. For the particles with 2rn ~< 20 nm Sepharose 4B offers better resolution but for
285
bigger particles as well as for studies of coagulation, flocculation, or aggregation the Sephacryl S 1000 column could be used. ACKNOWLEDGMENTS The author is indebted to Dr. B. Dr~aj for discussions and Mrs. I. Bu~ek for excellent technical assistance. REFERENCES 1. Nozaki, Y., Lasi~, D. D., Tanford, C., and Reynolds, J. A., Science (Washington, D. C.) 217, 366 (1982). 2. Pertofi, H., Laurent, T. C., Lilts, T., and K~gedal, L., Anal. Biochem. 88, 271 (1978). 3. Coil, H., and Fague, G. R., J. Colloid Interface Sci. 76, 116 (1980). 4. Granath, K. A., and Kvist, E. B., J. Chromatogr. 28, 69 (1967). 5. Ackers, G. K., J. Biol. Chem. 242, 3237 (1967). 6. Brunner, J., Skrabal, P., and Hauser, H., Biochim. Biophys. Acta 455, 322 (1976). 7. Reynolds, J. A., Nozaki, Y., and Tanf0rd, C., Anal. Biochem. 130, 471 (1983). 8. Perevucnik, S., Schurtenberger, P., Lasi~, D. D., and Hauser, H., Biochim. Biophys. Acta, 821, 169 (1985). 9. Fischer, T. H., and Lasi~, D. D., Mol. Cryst. Liq. Cryst. Lett. 102, 141 (1984). DANILO D. LASI(~
B. Kidri? Institute of Chemistry Ljubljana, Yugoslavia Received March 19, 1985
Journal of Colloid and Interface Science, Vol. 110,No. 1, March 1986
total volume, respectively, the value of (1 - K) is approaching the value of 1 where erf-l(1 - K) is rather independent of the argument.) By the use of a novel gel exclusion medium, Sephacryl S 1000, even bigger particles can be analyzed because between V0 (where the particles with 2rn >~ 300 nm elute) and Ft (with 2rn ~< 70 nm) all particles with sizes in this range are separated. Recently, this gel was successfully applied to vesicles where the diameter ranges between several dozen and several hundred nanometers (1). In this study we wanted to determine the size and size distribution of the silica sol particles by size exclusion chromatography using Sephacryl S 1000 and Sepharose 4 B CL gels. The columns were packed at a flow rate 4 ml/ h. After packing they were run with the elution solvent (0.01 MNaCI with NaOH added to pH = 9) for 2 days. For the columns 40 X 0.7 cm approximately ¼- ½ ml of 10 wt% silica sols (diluted immediately before the experiment) were chromatographed with a flow rate of 6 ml/h. The elution profiles were analyzed in fractions of ½- 1 ml by turbidimetry at 340 nm or by dry weight determination. Fast qualitative (but very sensitive) analysis was also made by the naked eye investigation of the Tyndall effect using
1
0
.
200
0
150
2O
15
/ J
IE
u.=
~
/// // o
100
O -
,.=, 10..~,
i/t3
.¢z c=l
c==
5O
'II
v
I
0.5
I
1
err-l(1 - g ]
1.0
1
I
{{
1.5
FIG. 1. Calibration curves of Sephacryl S 1000 (©) and Sepharose 4B CL ([3) columns. The equilibrium constant for the distribution of particles between the gel pores and fluid phase, K = (Ve - Vo)/(Ft - 1Io) w a s calculated from the positions of peaks of standards: for Sepharose column myoglobin (rH = 2 nm), aldolase (4.8 nm), ferritin (6.1 nm), and thyroglobulin (8.5 rim) were used while Sephacryl was calibrated with aldolase, ferritin, thyroglobulin, Ludox WP (rn = 1.05 nm) and latex beads (42.5, 50.5, and 110 nm coated with SDS).
282 0021-9797/86 $3.00 Copyright © 1986 by Academic Press, Inc. All fights of reproduction in any form reserved.
Journal of Colloid and Interface Science, Vol. 110, No. 1, March 1986
NOTES
:
V°
283
1
,
Vt
E c:
0.5
0.2 0.1 t,
B
B
10
12
14
lfi
EFFLUENT[g ]
FIG. 2. Elution profiles from Sephacryl S 1000 of different silica sols: Ludox TM (©), Tosil 1980 (Fq), and Tosil 1984 (A).
a laser beam (He-Ne, ~ = 632.8 nm). The Sephacryl S 1000 (Pharrnacia Fine Chemicals AG, Ztifich, Switzerland) column was calibrated with Dow Latex microspheres (Serva Feinbiochemica, Heidelberg, Federal Republic of Germany) with diameters 481 + 1.8, 364 + 2.4, 220 _+ 6.5, 109 _+ 2.7, and 85 _+ 5.5 nm with sodium dodecyl sulfate (SDS) added to eluent at a concentration of 0.1%. V0 was determined with 481-nm beads coated with SDS (the 364nm beads elute within experimental error at the same V=) while for Vt c ytochrome c, colored inorganic salts or complexes were used. Sepharose 4B CL (Pharmacia Fine Chemicals AB Uppsala, Sweden) was calibrated with 220and 85-rim beads (V0), cytochrome c and CuSO4 (Vt) and
with the proteins myoglobin, aldolase, ferritin and thyroglobulin, which were also used for the first column. The obtained standardization curves (5), 2rn(nm) = 37 erf-~(1 - K) for the Sepharose and 2rn(nm) = 155 err-l(1 - K) 35 for the Sephacryl are almost identical to the reported ones in the literature (6-8). Only in the region below erf-~(1 K) = 0.5 we approximated the standardization curve of Sephacryl column with the slightly concave exponential curve 2rn(nm) = 75 exp(3.6 eff-~(1 - K)). This curve is the best fit in the region where Ve ~ V0 and is similar to the reported one (7) except that it intercepts the ordinate closer to the real cut offdiameter. The calibration curves are shown in Fig. 1.
E (-,-)
OJ
.<
0.2
0.1
4
6
B
10
12
14 EFFLUENI[g]
FIG. 3. Elution profiles from Sepharose 4B CL of different silica sols: Tosil 1984 (A) and silica sol prepared without growth of particles (O).
Journal of Colloid and Interfac,e Science, Vol. 110, No. 1, March 1986
284
NOTES r=
l Vt
"~ 0.~ 2:
~ 0.3 N "~ Od
0.! ,
2
z,
G
8
10
12
14 EFFLUENT[g]
FIG. 4. Elution profiles from Sephacryl S 1000 of silica sols prepared with exaggerated (@) and without growth (©) of the particles and of aged Ludox HS (×).
Figure 2 shows some typical elution profiles of the standard silica sols on the Sephacryl S 1000 column. Average diameters of solutions of Ludox TM (O) (du Pont de Nemours, Wilmington, DEE), Tosil (n) (Chemapol, Prague, Czechoslovakia, produced in 1980), and Tosil (A) (1984) are 22.4, 21.3, and 14 nm, respectively. Because
these particles elute close to Vt they can be analyzed also by the Sepharose 4B, or Sepharose 2B (9), column and some typical elution profiles are shown in Fig. 3. The obtained diameters, 15.5 nm for Tosil 1984 (A) and 10 nm for the silica sol prepared in this laboratory (©) (see below) are in good agreement with the Sephacryl S 1000 results.
i,'
\\
///
I x\
~-.
0.20
"\
020
,
\ \
/
0.15 /
,~0.10
0.15
r
I-r--
..0=OlD
~
"~ 0.05
12
10
20
24
28
DIAMETER [ nm ]
32
6
10
14
18
22
DIAMETER [ nm ]
FIG. 5. Size distribution histograms (ndnt as a function of diameter, number of counted particles nt ~ 300) of (A) Tosil (1980) and (B) Tosil (1984) as determined by electron microscopy (samples were diluted to 0,1% and immediately deposited on carbon coated grids). The broken line represents the apparent size distribution of the chromatographic peaks. (Artificially, we applied the same procedure as was used for the peaks to all the points of the elution profile nevertheless the distribution coefficient K has physical meaning only at the equilibrium value, i.e., the top of the peak. This simplification also neglects the contribution of the diffusion and possible experimental parameters to the line broadening which is attributed solely to the superposition of the lines of heterogeneous sample (4). The resulting linewidth is at half-height of the ehition peak more than I5 nm and increases when V=approaches Vo.) These distribution curves indicate that in most cases the natural linewidth is too broad to allow the determination of the size distribution,
Journal of Colloid and Interface Science, Vol. 110, No. 1, March 1986
NOTES The elution profiles of some homemade silica sols on Sephacryl S 1000 column are shown in Fig. 4. The first one (O) was prepared without growth.of particles (to pH = 9 neutralized acidic silica sol was concentrated without heating at constant volume) while for the second one (O) the particles were grown (the diluted silica sol, pH = 9, was heated at constant volume for 2 h and concentrated to a very viscous sol which gelled overnight). The latter shows a very broad, heterogeneous profile with particles up to diameter of 175 nm while the former consists of very small particles (2rH = 9 nm, see also Fig. 3). The aged Ludox HS sample (×) also shows large particles (2rn = 166 nm). The results are in agreement with EM micrographs, In Fig. 5 the frequency distribution of the two Tosil samples is presented. The size of the particles determined by dectron microscopy and by size exclusion chromatography is in good agreement while the size distribution of the particles cannot be quantitatively determined because the natural linewidth of the elution peaks is more than ~ 15% of the column volume already for very homogeneous partides. Therefore, besides qualitative estimates from the widths of the peaks, these measurements can provide more evidence of the size distribution only for very heterogeneous samples (see also text of Fig. 5). The results indicate that size exclusion chromatography with commercially available gels is a simple and accurate method for the determination of the size of sol particles. Size distribution of sol particles can be determined only when the samples are very heterogeneous. However, due to the~slight changes in their properties during the usage, columns should be recalibrated after several runs if the quantitative analysis is desired. For the particles with 2rn ~< 20 nm Sepharose 4B offers better resolution but for
285
bigger particles as well as for studies of coagulation, flocculation, or aggregation the Sephacryl S 1000 column could be used. ACKNOWLEDGMENTS The author is indebted to Dr. B. Dr~aj for discussions and Mrs. I. Bu~ek for excellent technical assistance. REFERENCES 1. Nozaki, Y., Lasi~, D. D., Tanford, C., and Reynolds, J. A., Science (Washington, D. C.) 217, 366 (1982). 2. Pertofi, H., Laurent, T. C., Lilts, T., and K~gedal, L., Anal. Biochem. 88, 271 (1978). 3. Coil, H., and Fague, G. R., J. Colloid Interface Sci. 76, 116 (1980). 4. Granath, K. A., and Kvist, E. B., J. Chromatogr. 28, 69 (1967). 5. Ackers, G. K., J. Biol. Chem. 242, 3237 (1967). 6. Brunner, J., Skrabal, P., and Hauser, H., Biochim. Biophys. Acta 455, 322 (1976). 7. Reynolds, J. A., Nozaki, Y., and Tanf0rd, C., Anal. Biochem. 130, 471 (1983). 8. Perevucnik, S., Schurtenberger, P., Lasi~, D. D., and Hauser, H., Biochim. Biophys. Acta, 821, 169 (1985). 9. Fischer, T. H., and Lasi~, D. D., Mol. Cryst. Liq. Cryst. Lett. 102, 141 (1984). DANILO D. LASI(~
B. Kidri? Institute of Chemistry Ljubljana, Yugoslavia Received March 19, 1985
Journal of Colloid and Interface Science, Vol. 110,No. 1, March 1986