Bimodal chromatographic resolving zone

Disclosed herein is a resolving zone for a chromatograph comprising a plurality of porous, silica macroparticles chosen to provide the resolving zone with a bimodal pore distribution, the average pore size for each mode being such that the linear portions of the molecular weight calibration curve for each pore size in the bimodal distribution are nonoverlapping and the pore volume of each mode being such that the aforesaid linear portions are substantially parallel. The macroparticles can be either totally or superficially porous. The resolving zone can be composed of either a plurality of macroparticles each having a bimodal pore distribution or a plurality of macroparticles having one pore size distribution and a plurality of macroparticles having another pore distribution.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to chromatography, particularly size exclusion 
chromatography, and to the composition of the resolving zones used in such 
chromatography. It also relates to a process for performing 
chromatographic separation. 
2. Discussion of the Prior Art 
U.S. Pat. No. 3,505,785 discloses superficially porous microspheriods, 
having an average diameter in the range of 5 to 500 microns, which are 
composed of an impervious core coated with a multiplicity of monolayers of 
colloidal inorganic particles having an average size in the range of 0.005 
to 1.0 microns. U.S. Pat. No. 3,855,172 discloses microspheriods which are 
porous throughout and have an average diameter in the range of 0.5 to 20 
microns. They are composed of colloidal inorganic refractory particles 
having an average diameter in the range of 0.005 to 1.0 microns. 
The use of such microspheriods in chromatography, particularly 
size-exclusion chromatography is well-known. In such application, porous 
microspheroids are used as packings for the chromatograph resolving zone 
and functions to separate the components of a sample based on differences 
in hydrodynamic size of the components. The molecular weight (MW) of the 
components can be calculated as a function of the hydrodynamic size. A 
plot of the molecular weight fraction eluted at a given retention volume 
(V.sub.R) for a particular packing material reveals that for a given pore 
volume in the packing material, certain molecular weight fractions are 
totally excluded because of their large size and certain molecular weight 
fractions are totally permeating because of their small size. Between 
these two extremes is a range of molecular weight fractions that will be 
preferentially retarded by contact with the porous particles, and 
materials containing these molecular weight fractions can be fractionated 
by that particular packing material. 
The actual working relationship in size exclusion chromatography is the 
molecular weight calibration curve which is normally a logarithmic plot of 
the molecular weight versus the retention volume. Molecular weight 
calibration curves characteristically have a substantially linear portion 
so that the molecular weight of a retained fraction of a sample can be 
determined accurately if the retention volume for that particular 
molecular weight fraction occurs in the linear portion of the molecular 
weight calibration curve and less accurately if it occurs outside that 
linear range. Molecular weight calibration curves characteristically have 
linear portions that span approximately two decades in the log molecular 
weight scale for a single pore size. To obtain a calibration curve with a 
linear portion spanning more than two decades of the molecular weight 
scale, the tendency is to use a chromatographic resolving zone composed of 
many columns, each having different pore sizes. Specifically, as taught by 
"Know More About Your Polymer", a 1976 publication of Waters Associates, 
Milford, Mass., to expand the linear range of the molecular weight 
calibration curve so that materials containing a wide range of molecular 
weight fractions can be separated and detected, four or five columns are 
combined, each with molecular weight calibration curves whose linear 
portions overlap one another. 
Unfortunately, the range of expected linear molecular weight calibration 
does not occur. The linear portion of the molecular weight calibration 
curve for the combined particles can be increased in this way but the 
maximum appears to be only about three decades, which often is less than 
the range of molecular weight found in normal sample compositions. 
SUMMARY OF THE INVENTION 
According to this invention, there is provided a resolving zone for a 
chromatograph which provides a wider range of linearity in its molecular 
weight calibration curve and avoids the inconvenience of using multiple 
columns with overlapping linear calibration. This resolving zone comprises 
a plurality of porous macroparticles chosen to provide the resolving zone 
with a bimodal pore distribution, the average pore size for each mode 
being such that the linear portion of the molecular weight calibration 
curves for each pore size in the bimodal distribution are substantially 
nonoverlapping, the pore volume of each mode being such that the aforesaid 
linear portions are substantially parallel. As used herein, the term 
"average pore size" means volume average pore size. To achieve the maximum 
range of the linear portion of the molecular weight calibration curve, the 
pore sizes of the modes of the bimodal distribution should be about one 
order of magnitude apart. The term "bimodal pore distribution" as used 
herein is not meant to exclude the use of more than two modes of particle 
size distribution so long as two adjacent modes have substantially 
parallel linear non-overlapping portions of their calibration curves, the 
average pore volume of each of the two adjacent modes being separated 
about one order of magnitude. 
The macroparticles useful in this invention can be refractory particles 
such as silica or alumina or they can be non-refractory such as 
crosslinked polymer gels. In the preferred embodiment the macroparticles 
are refractory macroparticles composed primarily of silica and the 
component of the bimodal pore distribution having the smaller average pore 
size provides about 30 to about 60%, preferably about 40 to about 60%, 
more preferably about 40 to 55% and still more preferably, about 45 to 
55%, of the total pore volume of the macroparticles in the resolving zone 
with the balance of the total pore volume being provided by the component 
of the bimodal distribution having the larger average pore size. The 
resolving zone can be composed of a plurality of macroparticles, each 
having a bimodal pore distribution, or it can be composed of a plurality 
of macroparticles each having one pore distribution combined with a 
plurality of macroparticles having another pore distribution. 
This invention also provides an improved process for performing 
chromatographic separation comprising the steps of: 
(a) placing the material to be separated in a carrier fluid; 
(b) contacting the carrier fluid with a resolving zone comprising a 
plurality of porous, refractory, macroparticles chosen to provide the 
resolving zone with a bimodal pore distribution the average pore size for 
each mode being such that the linear portions of the molecular weight 
calibration curve for each pore size in the bimodal distribution are 
nonoverlapping, and the pore volume of each mode being such that the 
aforesaid linear portions are substantially parallel, and 
(c) determining the extent of retention of the materials by the resolving 
zone.

DETAILED DESCRIPTION OF THE INVENTION 
In size exclusion chromatography, a chromatograph such as that shown 
schematically in FIG. 3 is used. A material to be separated is injected 
into a carrier fluid stream at some injection point 12 and forced under 
pressure through a chromatographic resolving zone 13 to a detector 14. In 
passing through the resolving zone, the materials in the carrier fluid 
contact the packing material in the resolving zone and are retained for a 
time characteristic of their molecular weight (MW). In time, as more 
volume of carrier fluid passes through the chromatographic column, the 
material temporarily retained by the packing material is eluted from the 
column. The detector determines when each component of the material leaves 
the resolving zone. The output of the detector is characteristically a 
peak such as that shown schematically in the bottom of FIG. 4. 
The resolving zones used in size exclusion chromatography are generally 
columns packed with porous particles such as those described in U.S. Pat. 
No. 3,505,785 and U.S. Pat. No. 3,855,172, or more recently the 
macroporous microspheroids disclosed in U.S. Pat. No. 4,040,286 by R. K. 
Iler and J. J. Kirkland. 
Using such packing materials, a typical relationship of the log MW (a 
function of solute hydrodynamic radius) versus retention volume (V.sub.R) 
is shown by the single solid line on the left-hand side of FIG. 1. The 
limiting retention volume at a point A is known as the total exclusion 
volume, which is determined by the maximum pore size available for 
permeation by the solute materials that are totally rejected from the 
internal porosity elute at this retention volume, and solutes 
corresponding to this molecular weight and larger are not fractionated by 
the system. Point B represents the volume associated with species which 
totally permeate the internal pores of the packing material, and is known 
as the total permeation volume. Thus, materials corresponding to this MW 
and smaller cannot be substantially fractionated by this separating 
system. The difference between retention volumes A and B represents 
partial permeation of solutes, and it is within this volume range that 
separation occurs. The difference between the retention volume at B and 
retention volume at A is a function of the total internal pore volume of 
the packing. Between retention volumes at A and B there is an 
approximately linear region of the log MW versus retention volume curve 
(points C to D) which is described by the following equations: 
EQU V.sub.R =C.sub.1 -C.sub.2 logMW (1) 
and 
EQU MW=D.sub.1 e.sup.-D.sbsp.2.sup.V.sbsp.R (2) 
c.sub.2 is the slope of the linear portion of the calibration curve (in 
ml/decade-MW) and C.sub.1 is the intercept of this linear portion. To 
extract molecular weight information from this calibration plot, 
experimental chromatograms and equation 2 above are utilized. D.sub.1 
relates to the intercept of this linear portion of the calibration curve 
and D.sub.2 relates to its slope. These equations are well-known to those 
skilled in the art and are widely used by those characterizing 
macromolecules. 
The additive characteristics of two identical columns used in 
size-exclusion chromatography is well known. As indicated in FIG. 1, 
connecting two identical columns (same particles, same length) with 
identical calibration curves indicated by the two solid lines is 
equivalent to doubling the length of a single column. As indicated, 
connecting these two columns increases the total available pore volume, 
thus increasing the retention volume range between total permeation and 
total exclusion, but maintaining the same molecular weight fraction range. 
As shown in FIG. 1 when two columns are connected, the molecular weight 
fraction range remains about 3,000 to 80,000, even though the retention 
volume is doubled. The calibration curve for the combination of the two 
columns is shown by the dashed line. The additive function describing this 
relationship is: 
##EQU1## 
Traditionally, polymer fractionation has been accomplished with packings 
having the broadest possible pore-size distribution. This is normally 
obtained by connecting several columns of different pore size to produce a 
separating system covering the molecular weight range of interest. FIG. 2 
shows a series of molecular weight calibration curves for six different 
chromatographic resolving zone, each filled with porous silica particles 
having different pore sizes. The designation and average pore volume for 
these six particles is given in Table I below: 
TABLE I 
______________________________________ 
Designation Pore Size (A.degree.) 
______________________________________ 
1 PMS-50 60 
2 PMS-300 125 
3 PMS-600 195 
4 PMS-800 300 
5 PMS-1500 750 
6 PMS-4000 3500 
______________________________________ 
These particles were made as described in U.S. Patent Application Ser. No. 
639,111, filed June 15, 1976 and U.S. Pat. No. 3,782,075. 
The bar graphs to the right of FIG. 2 indicate the linear range of each 
calibration plot. To achieve a linear combined calibration curve spanning 
a molecular weight range from 10.sup.3 to 10.sup.6, a combination of six 
columns traditionally would be used, each composed of the individual 
particles corresponding to the six graphs. 
In this invention the relationship given in equation 3 has been exploited 
to improve the accuracy, versatility, and convenience of the 
size-exclusion process. This relationship predicts a previously 
unrecognized phenomena, namely, that to obtain a wide linear log 
MW-retention volume relationship, a series of columns having substantially 
overlapping linear molecular weight fractionation ranges (i.e., linear 
portions) should not be used. Rather, columns having only two pore sizes, 
chosen so that the linear portions of the molecular weight versus 
retention volume curves do not overlap, should be used. This produces a 
far wider linear range in the calibration curve. As shown representatively 
in FIG. 4, a polymodal pore distribution in the resolving zone produces a 
narrow linear portion on the molecular weight calibration curve, and a 
bimodal distribution produces a much wider linear portion. The calibration 
curve for the polymodal distribution does not encompass the entire 
molecular weight distribution of the sample within its linear range, 
whereas the calibration curve for the bimodal distribution does. 
The advantage of using chromatographic columns having a bimodal pore 
distribution, whether connecting columns of individual pore size or using 
columns containing a physical mixture of particles that are two pore sizes 
can, therefore, be seen from FIG. 4. Molecular weight calibration curves 
of the type shown by the bimodal pore size distribution is greatly 
preferred when attempting to characterize a polymer with the type of 
molecular weight distribution illustrated at the bottom of the plot. 
A quantitative comparison of a polymodal pore-size distribution versus 
bimodal pore-distribution system is given in FIG. 9. FIG. 9 shows a 
polystyrene calibration curve for a polymodal and for a bimodal resolving 
zone. The set of columns used to produce the polymodal distribution are 
filled with a packing material labeled 1, 2, 4, 5 and 6 in FIG. 2. The 
individual columns have substantially overlapping calibration plots as in 
the traditional mode. The approximate linear calibration range (dashed 
line is the linear fit) of this combined broad pore-size distribution set 
is only about two and one-half decades of molecular weight. On the other 
hand, the bimodal distribution shown in FIG. 9, obtained by connecting 
columns of only two pore sizes (that of particles 1 and 5 in FIG. 2) 
results in a linear molecular weight calibration curve spanning more than 
four decades of molecular weight. 
To obtain these unexpected and improved results, the individual calibration 
curves for the two pore sizes used in the bimodal distribution must not 
overlap. This is achieved by choosing particles with the appropriate pore 
size. The average pore sizes of the bimodal distribution should be about 
one order of magnitude apart. With this bimodal approach, linear 
calibration curves having up to five decades of molecular weight range are 
obtained. A trimodal arrangement of similar type could result in up to 
seven decades molecular weight range linearity. In addition to having 
molecular weight calibration curves which are non-overlapping, the 
internal pore volume of the two modes should be such that the linear 
portions of the calibration curves are substantially parallel. The term 
substantially parallel means that the shapes of the linear portions of the 
calibration curves need not be exactly parallel provided some deviation 
from linearity can be accepted. For example, reference to FIG. 9 indicates 
that the overlapping polymodal calibration curve which represents the 
prior art is far from linear over the range predicted. When about 30 to 
60%, preferably 40 to about 60%, more preferably about 40 to 55% and still 
more preferably about 45 to 55%, of the total pore volume of the 
macroparticles in each resolving zone is provided by the component of the 
bimodal pore distribution having the lower average pore size with the 
balance provided by the component having the larger average pore size, the 
linear portions of the individual calibration curves are substantially 
parallel. Reference to FIG. 10 shows the deviation from linearity in the 
calibration curve when the pore volume ratio is 40:60. In the most 
preferred embodiment, however, each component of the bimodal pore 
distribution should provide about 50% of the total pore volume of the 
macroparticle in the resolving zone to reduce deviation from linearity in 
the calibration curve. 
Best results are obtained using packing materials with a very narrow 
pore-size distribution. Pore size distributions of each of the bimodal 
systems should be 1.0 or less (2.delta.) as shown in conventional 
log-normal plots of mercury porosimetry measurements. Ranges of 0.5 
(2.delta.) are preferred. Within these ranges, pore size distribution is 
an insignificant factor in determining the D.sub.2 of the calibration 
plot, and the internal volume of the particles is dominant in determining 
the D.sub.2 of the calibration plot. If the pore size distribution is 
larger than the values given above, then the interrelationship of both 
pore size distribution plus internal volume determines the slope of the 
calibration curve. 
The bimodal pore distribution used in the present invention can be achieved 
in one of two ways. The bimodal pore distribution can be provided by a 
plurality of microparticles each having a bimodal pore distribution. In 
this instance, a single column such as that shown in the upper portion of 
FIG. 3 can be used. Alternately the bimodal pore distribution can be 
provided by using a plurality of macroparticles having one pore 
distribution and a plurality of macroparticles having another pore 
distribution. While particles with different pore size distributions can 
be mixed into one column, the packing of such columns is less convenient 
and it is best to use two or more columns, each packed with a single type 
particle. 
Individual particles of the desired pore size to produce the bimodal pore 
distribution can be produced by the techniques described in the patents 
and the patent applications mentioned above. The disclosure in these 
patents and applications is hereby incorporated by reference into the 
present specification. Polymeric gels, alumina and the wide range of 
refractory particles mentioned in these documents can be used, but silica 
is the preferred material, particularly for chromatographic separations. 
Particles having a bimodal pore distribution can either be totally porous 
or superficially porous macroparticles. The term macroparticle, as used 
herein, means the composite macroparticle (either totally or superficially 
porous) having an average diameter in the range of about 0.5 to about 500 
microns. The totally porous embodiment of this particle is shown in FIG. 
5. Here the macroparticle 15 has an average diameter of about 0.5 to 500 
microns. Preferred macroparticles having average diameters of about 5 to 
50 microns. The macroparticle is composed of a plurality of microparticles 
16, each having an average diameter in the range of about 0.005 to about 
1.0, preferably about 0.005 to 0.5, microns. The individual microparticles 
are in turn composed of a plurality of ultramicroparticles 17 having an 
average diameter in the range of about 1.0 to about 30.0 nanometers with 
2-20 being preferred. Between each microparticle is a macropore 18, and 
between each ultramicroparticle is a micropore 19. While these particles 
can in general have any shape, it is preferred that they have a spherical 
shape so that the macroparticles are actually macrospheres, the 
microparticles are microspheres and the ultramicroparticles are 
ultramicrospheres. The spherical nature of these materials improves their 
performance in chromatographic columns. 
Alternatively, as shown in FIG. 6, the totally porous macroparticle 15 can 
be composed of a core 20 comprising a plurality of ultramicroparticle 21 
having an average diameter in the range of about 1 to about 30 nanometers, 
and a skin composed of a multiplicity of microparticles 22, each having a 
diameter in the range of about 0.1 to about 1.0 microns, or more commonly, 
0.1 to 0.5 microns. The totally porous macroparticle produced by currently 
known techniques preferably have a diameter in the range of about 0.5 to 
about 50 microns. 
One embodiment of a superficially porous macroparticle is shown in FIG. 7. 
Such macroparticle 22 has a diameter in the range of about 0.5 to about 
500 or preferably, 5 to 50, microns and comprise an impervious macrocore 
24 and a coating of a multiplicity of like monolayers of like colloidal 
inorganic microparticles 25 joined to and surrounding the core. Each 
microparticle has an average diameter in the range of about 0.005 to about 
1.0 microns or preferably, 0.1 to 0.5 microns and comprises from about 0.2 
to about 25% of the total volume of the macroparticle. The microparticle 
can be similar to that shown in FIG. 5, composed totally of 
ultramicroparticle, or it can be similar to the microparticle shown in 
FIG. 7, composed of an impervious microcore 27 and a coating of a 
multiplicity of like monolayers of like colloidal inorganic 
ultramicroparticles 28 joined to and surrounding the core. 
In either case, for the totally porous or the superficially porous 
particles, the pores between the individual microparticles in the 
macroparticle shall be referred to as the macropore 30 and provides one 
mode of the bimodal pore distribution, and the pores between the 
individual ultramicroparticle shall be referred to as the micropore 31 and 
provides the other mode of the bimodal pore distribution. Recent 
terminology sometimes defines pores of the size designated as "micropores" 
herein as "mesopores". 
EXAMPLE I 
The following describes the preparation of pellicular particles with a 
bimodal pore-size distribution. Such a structure is shown in FIG. 7. 
75 g of Zipax.RTM. (Du Pont trademark for chromatographic support) 
controlled porosity support (E. I. du Pont de Nemours and Co.) (&lt;37 .mu.m) 
was stirred gently with 800 ml of 0.5% Lakeseal laboratory cleaner 
solution for 30 minutes. The excess solution was removed by decantation 
and washed with distilled water. This operation was repeated seven times, 
and the resulting powder filtered on a coarse sintered-glass filter and 
dried in air. The dry powder was then placed in a three-inch diameter 
coarse sintered-glass funnel and treated with 100 ml of 0.5% Zelex.RTM. DX 
(Du Pont trademark for antistatic agents and mold release agents) (E. I. 
du Pont de Nemours and Co.) solution for five minutes with stirring. The 
treated beads were filtered, then washed twice with 200 ml of distilled 
water and dried in the funnel with vacuum. 
The beads then were treated with 100 ml of 10% silica sol made from 
Ludox.RTM. AS (Du Pont trademark for colloidal silica) (.about.140 A 
silica particles supplied by E. I. du Pont de Nemours and Co.) (125 g of 
30% by weight silica in Ludox.RTM. AS diluted to 400 g with distilled 
water). The mixture of beads and silica sol was allowed to stand for 15 
minutes in the funnel with frequent gentle stirring. Excess Ludox.RTM. was 
then filtered off and the resulting wet cake washed four times by gently 
slurrying with about 400 ml of tap water and filtering. The cake was then 
allowed to air-dry in the filter under vacuum. This material was then 
dried at 150.degree. C. for one hour in a circulating air oven and a small 
sample removed for surface area measurement. 
The Zelec.RTM. DX silica sol treatment described above was repeated three 
more times to build up a crust of the 140 A silica sol ultramicroparticles 
on the surface of the 2000 A silica microparticles which originally made 
up the crust of the Zipax.RTM. particles. The final particles were dried, 
and heated at 650.degree. C. for two hours to burn out the organic 
interlayer and sinter the particles into a mechanically stable condition. 
This sintered sample was then allowed to stand for two hours in a large 
excess of 0.001 M ammonium hydroxide with frequent stirring. The particles 
were then washed twice with a large excess of distilled water by 
decantation, filtered on coarse sintered-glass funnel, air-dried, and 
heated at 150.degree. C. for two hours in a circulating air bath. The 
final material was dry-sieved with stainless screens to obtain a &lt;38 .mu.m 
fraction of 45 g. 
Surface areas on the products obtained during the synthetic steps were 
obtained by the nitrogen flow method with the following results: 
______________________________________ 
Sample Surface Area, m.sup.2 /g 
______________________________________ 
Starting Zipax.RTM. 
0.89, 0.99 
First treatment with 
Ludox.RTM. AS 2.03, 2.09 
Second treatment with 
Ludox.RTM. AS 2.35, 2.46 
Third treatment with 
Ludox.RTM. AS 3.07, 3.01 
Fourth treatment with 
Ludox.RTM. AS 3.38, 3.50 
Sintered at 650.degree. C. for 
two hours 2.67, 2.67 
Final rehydrated material 
2.85, 2.86 
______________________________________ 
A mercury porosimetry measurement of this sample showed three breaks in the 
mercury intrusion plot, one at about 10 microns, representing the 
intrusion of mercury between the individual particles, a break at about 
0.07.mu. (700 A) representing the macropores between the sol 
microparticles in the crust of the initial Zipax.RTM. structure, and a 
break at about 0.006.mu. (60 A) representing the pores between the 140 A 
sol ultramicroparticles which are multilayered onto the original 
Zipax.RTM. structure by the procedure herein described. The volumes 
associated with the bimodal pore-size distribution were: 
Macropores--(700 A pores)--0.011 cc/g 
Micropores--(60 A pores)--0.014 cc/g. 
These data show that the final particles contained the desired bimodal pore 
configuration, with pores approximately one decade in size difference, and 
approximately equal pore volumes for each pore size. 
EXAMPLE II 
Particles of the type illustrated in FIG. 6 can be prepared as follows: 15 
g of porous silica microspheres (PSM-40; 47 angstrom pores) made according 
to U.S. Pat. No. 3,782,075, Jan. 1, 1974, Joseph J. Kirkland, assigned to 
Du Pont; U.S. Pat. No. 3,855,172, Dec. 17, 1974, Ralph K. Iler and Herbert 
J. McQueston, assigned to E. I. du Pont de Nemours and Co., was treated 
with 200 ml of 0.001 M ammonium hydroxide, allowed to stand for 10 minutes 
with occasional stirring and centrifuged in a 250 ml polyethylene bottle 
for two minutes (from the start) at approximately 2,000 revolutions/min. 
The clear supernatant was decanted, and to the wet cake was added 100 ml 
of 0.5% Zelec.RTM. DX (E. I. du Pont de Nemours and Co.) solution which 
had been adjusted to pH 7 with ammonium hydroxide. The sytem was carefully 
slurried, then left to stand for 10 minutes with occasional gentle 
stirring. The resulting mixture was centrifuged for one minute using the 
approach described above, and the excess Zelec.RTM. DX solution decanted. 
The wet cake was washed twice with 200 ml of distilled water (adjusted to 
pH 7 with ammonium hydroxide) by carefully slurrying, centrifuging for one 
minute, and decanting. 
To these treated particles was added 50 ml of 5% (by weight) 2000 A silica 
sol mixture adjusted to pH 8 [sol can be prepared by procedures in: W. 
Stober, A. Fink and E. Bohn, J. Colloid, Inter. Sci., 26, 62 (1968)], and 
the mixture was thoroughly slurried and occasionally stirred for 10 
minutes. This mixture was centrifuged as above, decanted and excess sol 
retained. The coated beads were then washed twice with 200 ml of distilled 
water (pH 7) by slurrying, centrifuging and decanting. The second wash 
decantant from this process was clear. The wet cake was then filtered on a 
3 .mu.m "Nuclepore" filter and dried in a circulating air oven at 
150.degree. C. for two hours. The sample was then fired at 700.degree. C. 
for one hour in a muffle furnace. A small portion of this material was 
subjected to scanning electron micron analysis which showed an excellent 
coverage of the surface of the original beads with the 2000 A silica sol. 
No bare spots were seen on the particles. 
A second layer of 2000 A silica sol was placed on the PSM particles using 
the technique described above, after first hydrolyzing the fired silica 
particles in 0.01 M hydrochloric acid overnight and eliminating the acid 
by washing with distilled water. The material was again treated with 
Zelec.RTM. DX, followed by 2000 A sol (25 ml of "virgin" plus the 
recovered sol excess from the first treatment), in the manner described 
above. Inspection of this material by scanning electron microscopy showed 
the second coating was layered as desired. Very few spots were seen on the 
beads, and only a very small amount of particle bridging was noted. 
A third, fourth, fifth, and sixth treatment of the beads were carried out 
in essentially the same manner as described above to build up the desired 
crust of 2000 A silica sol particles on the particles. These treated beads 
were fired at 750.degree. C. for one hour and rehydrolyzed by dilute acid 
treatment as above. SEM inspection of the final beads showed good 
coverage, but it was not possible to observe the exact thickness of the 
desired superficially porous crust. 
Mercury intrusion measurements plotted in FIG. 8 show that the pore volume 
of the larger pores of these particles is about 40% of the total pore 
volume and the pore volume of the smaller pore volume is about 60% of the 
total. The log molecular weight versus retention volume calibration plot 
for a 25.times.0.62 cm i.d. column of these particles is shown in FIG. 10. 
Because of the difference in internal volumes associated with the two 
modes, there is some deviation from linearity.