Pellicular coated support and method

A pellicular coated support and method for producing such a support are disclosed. The support includes a thin layer of adsorbate that is adsorbed to and cross-linked on an inorganic support material with the thus formed support being particularly well suited for use in liquid chromatography. An inorganic support of silica, alumina or titania has the pellicular coating formed thereon with the coating being an amine that is crosslinked by a crosslinking agent of epoxy resin, bromide or nitro alcohol.

FIELD OF THE INVENTION 
This invention relates to a pellicular coated support and method for 
forming the same and, more particularly, relates to an ion-exchange 
support having a thin coating thereon that is particularly well suited for 
liquid chromatography. 
BACKGROUND OF THE INVENTION 
In modern high performance liquid chromotography (HPLC), pressures of 
several thousand lb/in.sup.2 are often developed within the chromatography 
columns. This requires that the column packing materials be rigid and 
non-collapsible. To achieve this, porous inorganic materials, such as 
silica and alumina, have heretofore been utilized as the support material 
with organic stationary phases on the surface to obtain a variety of 
liquid chromatography column packing materials. 
Previously prepared porous chromatographic packings with a stationary 
organic phase have used covalent bonding to attach the organic phase to 
the support surface. The coupling of an organic phase (P) to the surface 
of an inorganic support has been achieved through the use of an 
intermediate silane coupling agent wherein the silicone portion of the 
molecule is bonded to the support and the organic portion of the 
organosilane either is the organic phase or is attached to the organic 
phase. Such inorganic supports are shown, by way of example, in U.S. Pat. 
Nos. 3,983,299 and 4,029,583. 
The primary function of the coupling agent is to provide a chemical bond 
between the inorganic support and the stationary organic phase (P) that is 
responsible for the chromatographic partitioning. The general chemical 
formula for this composite may be expressed as: 
##STR1## 
where .tbd.Si indicates that three of the bonds on silica are bonded 
directly to the inorganic support or to adjacent organosilanes that are 
bonded to the support, --CH.sub.2).sub.m is a coupling arm that ties the 
rest of the stationary phase (P) to the support, and m is a number ranging 
from 0 to 18. 
In the preparation of chromatography packings it is necessary to strive for 
exact reproduction of the organic coating from batch to batch. It has been 
observed, however, that when an inorganic support is treated with a 
solution of organosilane it is difficult to reproducibly control the 
amount of organosilane that is deposited on the surface, particularly 
since many ways have been developed whereby organosilanes may be bonded to 
an inorganic surface. Chromatographically, the amount of organosilane 
deposited on the surface of an inorganic support is quite important. The 
chromatographic behavior of a bonded phase packing material is totally 
dependent on the nature and amount of organic material on the particle. 
A further disadvantage of the organosilane bonding chemistry is that the 
bonded phase has been found to erode. As columns are operated and hundreds 
of column volumes of mobile phase pass through the column, organosilane 
hydrolysis can occur as follows: 
##STR2## 
Although this reaction can be quite slow, it can still produce a 
substantial degradation in a relatively short period of time, as for 
example, in one month of continuous operation. 
SUMMARY OF THE INVENTION 
This invention provides a pellicular coating on a support material and a 
method for producing the same. A thin layer of organic adsorbate is 
adsorbed to and cross-linked on the surface of the inorganic support 
material with the thus formed support being particularly well suited for 
use in liquid chromatography with the adsorbed layer being the stationary 
phase. By use of this invention, the coated support is reproducible with 
controlled and uniform coating thickness and the thus provided support is 
stable in operation. 
It is therefore an object of this invention to provide an improved support 
having a pellicular coating thereon. 
It is another object of this invention to produce an improved support 
having a pellicular coating thereon that is stable and reproducible. 
It is yet another object of this invention to produce an improved 
chromatographic support. 
It is still another object of this invention to produce an improved support 
having a thin layer on support material with the layer being adsorbed to 
and crosslinked on the surface of said support material. 
It is yet another object of this invention to provide an improved packing 
material that includes an inorganic support material having an adsorbate 
adsorbed to and crosslinked on the surface of said support material. 
It is still another object of this invention to provide an improved process 
for producing a pellicular coating on a support material. 
It is yet another object of this invention to provide an improved process 
for adsorbing a thin layer to a support material and crosslinking the 
layer thus formed on said support material. 
It is still another object of this invention to provide an improved process 
for providing a pellicular layer on a support material by contacting the 
support material with an adsorbate so that the adsorbate is adsorbed on 
the surface of the support material to form a coating thereon that is 
crosslinked by exposure to a crosslinking agent. 
With these and other objects in view which will become apparent to one 
skilled in the art as the description proceeds, this invention resides in 
the novel support apparatus and method for forming the same substantially 
as hereinafter described, and more particularly defined by the appended 
claims, it being understood that such changes in the precise embodiment of 
the herein disclosed invention are meant to be included as come within the 
scope of the claims.

DESCRIPTION OF THE INVENTION 
This invention is particularly directed toward achieving an adsorbate layer 
on a support material that is reproducible with respect to thickness and 
uniformity as well as achieving stability during operation for the thus 
produced support or packing material. 
It is widely accepted in physical chemistry that the concentration of a 
solute at a liquid-solid interface can be greater than that in the bulk 
solution. This adsorption onto surfaces may be expressed by the equation: 
##EQU1## 
where S is the excess concentration of solute/cm.sup.2 of surface, dr/dc 
is the rate of increase of surface tension of the solution with the 
concentration of the solute, R is the gas constant, C is solute 
concentration to solution, and T is absolute temperature. Any substance 
which decreases the surface tension at an interface will concentrate at 
that interface. 
In the event that this adsorption from solution leads to the formation of a 
single layer of solute molecules on the surface of the solid, the equation 
a=kC.sup.n provides an adequate description of the adsorption process over 
a wide range of concentrations where a is the amount of solute adsorbed by 
a unit mass of adsorbent, C is solute concentration in the solution, and 
the terms k and n are constants for a given adsorbent and adsorbate. 
By controlling the polarity of the solvent the adsorption of polar solutes 
onto surfaces can be controlled. The less polar the solvent, the stronger 
the adsorption. With a polarized surface (designated as 
P.sub.s.sup..delta.-) and a solvent (designated as P.sub.m.sup..delta.+), 
then the adsorption of solute (designated as S.sup..delta.+) to the 
surface may be described as follows: 
EQU P.sub.s.sup..delta.- +S.sup..delta.+ .revreaction.P.sub.s.sup..delta.- 
S.sup..delta.+ 
while the adsorption of polar solvent may be described by the equation: 
EQU P.sub.s.sup..delta.- +P.sub.m.sup..delta.+ 
.revreaction.P.sub.s.sup..delta.- P.sub.m.sup..delta.+ 
Both P.sub.m.sup..delta.+ and S.sup..delta.+ are obviously competing for 
the surface P.sub.s.sup..delta.-. The ultimate composition on a surface 
depends on the relative affinities of the different components for the 
surface and their concentrations. 
When the affinity of an organic solute (designated as R) is sufficiently 
large, the surface will become saturated with a layer of organic molecules 
R ranging from one to a few molecules thick. This technique provides a 
convenient method for organizing molecular films on a surface that are 
very thin and uniform. Accumulation of organic molecules R on the surface 
is self-limiting; when all active sites on the surface are covered, 
adsorption ceases. These thin, reproducible films are highly desirable in 
both the preparation and use of chromatography supports. 
It is apparent, however, that an adsorbed organic layer might not have 
adequate long-range stability in a chromatographic column when the support 
is eluted with thousands of column volumes of solvent. A gradual leaching 
of even the most tenaciously adsorbed solutes would occur. 
In this invention, adsorption is used to establish films of organic 
molecules on surfaces followed by stabilization of this film through 
covalent crosslinking of adjacent molecules into a continuous surface skin 
or layer. By this crosslinking process, the solubility of the adsorbed 
films of organic molecules is substantially decreased and the number of 
adsorption sites at which desorption must occur simultaneously to elute 
the coating is greatly increased. 
Preparation of adsorbed organic phase supports has been achieved by a 
synthetic route in which the organic phase is adsorbed directly to the 
surface of native inorganic support materials. Adsorption may occur 
through ionic forces, van der Waals forces, and/or hydrogen bonding 
between organic functional groups and the surface. To prevent this 
adsorbed layer from eluting, adjacent organic groups are crosslinked. 
These supports will be referred to as simple adsorbed and crosslinked 
phases. 
In simple adsorbed and crosslinked phases, adsorption of a surface 
monolayer of organic compound may be represented schematically as follows: 
##STR3## 
where A is a site on the substrate surface that has an affinity for an 
organic adsorbate, Y is a functional group on the organic adsorbate that 
interacts with A to cause the adsorption of the whole organic moiety to 
the surface, R is an organic compound as described above, and X is a 
functional group on R that is used to crosslink adjacent adsorbed species. 
It is important that each adsorbate be capable of participating in at least 
two crosslinking reactions to produce high molecular weight pellicular 
layers. In a general model, the adsorbate may be represented as: 
EQU (Y).sub.m --R--(X).sub.m 
where Y and X may be the same or different functional groups and m and n 
may vary from 1 to several thousand. 
Crosslinking of adjacent adsorbate molecules was achieved with a 
crosslinker described by the general formula: 
##STR4## 
A and B are functional groups on the crosslinker molecule G that react with 
X on the adsorbate to produce chemical bonds that couple adsorbate 
molecules together on the surface. Species A and B may be the same or 
different functional groups while m and n may vary from 1 to several 
hundred. On occasion it may be desirable to further modify the properties 
of the organic stationary phase to facilitate its use in chromatography. 
By using a crosslinking agent G that has additional organic moieties C and 
D covalently bonded, it is possible to change the chromatographic 
properties of a packing without changing all of the bonding chemistry. The 
number of C and D ligands represented by o and p may vary from zero to 
several hundred in a crosslinking molecule. 
EXAMPLE 1 
Preparation of a Polyethyleneimine Coating on Porous Silica 
4 grams of LiChrospher Si 500 (10 micron particle diameter) were swirled in 
a solution of 1.5 grams of polyethyleneimine 6 (average molecular 
weight=600) in 15 ml of methanol. Air was removed from the pores of the 
support with a light vacuum. The silica was collected by filtration and 
air-dried for 40 minutes on a reduced-pressure funnel. The coated silica 
was again swirled and degassed in a crosslinking solution, which consisted 
of 1.25 grams of pentaerythritol tetraglycidyl ether dissolved in 12.5 ml 
of dioxane. The mixture was left for 16 hours at room temperature, then 
heated on a steambath for 40 minutes with swirling at 10-minute intervals. 
The product was collected by filtration and washed several times each with 
acetone, water, and again acetone, then air dried. 
The resulting coated silica was assayed for its capacity to bind and 
release both small molecules (picric acid) and macromolecules (hemoglobin) 
in solution. the ion-exchange capacity (IEC) for picric acid was 1.32 
millimoles per gram of coated support while that for hemoglobin was 65 
milligrams per gram of coated support. 
Elemental analysis of the coated silica showed 5.59% C, 1.29% H and 2.26% 
N. When the oxygen (approximately 1.3%) is included, it is seen that the 
coating makes up 10% of the mass of the product. The ratio of C to N 
suggests that 21% of the nitrogen residues are crosslinked. Comparing the 
IEC for picric acid and elemental analysis shows that 77% of the nitrogen 
residues were detected by the picric acid assay and thus can participate 
in ion-exchange. 
250 milligrams of coated support were suspended in 10 ml of 1 molar NaCl 
and titrated with 0.1 normal HCl. The resulting change in pH of the 
suspension, as shown in FIG. 1, illustrates that the material functions as 
a weak anion exchanger, with charge decreasing in a continuum over the pH 
range 0-9. As shown in FIG. 1 of titration curves in 1 M NaCl, A is 
LiChrosorb Si 100 (10-micron particle diameter), 0.25 g and B is 
LiChrosorb Si 100 (10-micron particle diameter) with a coating of 
polyethyleneimine 6: 0.25 g with an IEC=495 micromoles of picric acid. 
Polyethyleneimine-coated silica was packed into 4.2 mm.times.25 cm columns 
in the form of a slurry. Columns packed with coated large-pore silicas 
(LiChrospher Si 500; 10-micron particle diameter) were used to resolve 
mixtures of proteins. Resolution of human serum proteins is shown in FIG. 
2. In FIG. 2, the column was polyethyleneimine-coated LiChrospher Si 500 
(10-micron particle diameter), 25.times.0.4 cm; the sample was 100 .mu.l 
of human serum, diluted 33%; the eluent was 20 min linear gradient; 0.02 M 
Trisacetate, pH 8.0, to 0.02 M Tris-acetate+0.5 M sodium acetate, pH 8.0; 
the flow rate was 2.0 ml/min.; the inlet pressure was 1400 psi and the 
detection was A.sub.280 monitored. 
Isoenzymes were detected by monitoring their enzymatic activity with a 
suitable post-column reactor and detector; FIG. 3 shows the activity 
profile of lactate dehydrogenase isoenzymes from a rat kidney homogenate. 
In FIG. 3 the column was polyethyleneimine-coated LiChrospher Si 500 
(10-micron particle diameter), 25.times.0.4 cm.; the sample was 100 .mu.l 
of 105,000.times.g supernatant, diluted 15.times.; the flow rate was 1.5 
ml/min; the inlet pressure was 2600 psi and the detection was post-column 
reactor with lactic acid and NAD; fluorescence monitored. 
Silicas with a higher surface area (LiChrosorb or LiChrospher Si 100; 
10-micron particle diameter) were coated, packed into columns and used to 
resolve mixtures of small molecules. The high IEC and selectivity of this 
packing permit isocratic resolution of many compounds such as carboxylic 
acids, as shown in FIG. 4, and phenols, as shown in FIG. 5. 
In FIG. 4, phenoxyacetic acids are shown as follows: A) Phenoxyacetic acid; 
B) o-Chlorophenoxyacetic acid; C) 2,6-Dichlorophenoyacetic acid; D) 
2,4-Dichlorophenoxyacetic acid; E) 2,3-Dichlorophenoxyacetic acid and F) 
2,4,5- Trichlorophenoxyacetic acid, with the column being 
polyethyleneimine-coated LiChrosorb Si 100 (10-micron particle diameter), 
25.times.0.4 cm.; the sample being 100 .mu.l containing 0.02 mg each 
compound; the eluent being 0.05 M potassium phosphate+0.1 M sodium 
acetate, pH 7.5; the flow rate being 1.0 ml/min; the inlet pressure being 
1250 psi and the detection being A.sub.254 monitored. 
In FIG. 5, phenols are shown as follows: A) Phenol; B) Catechol; C) 
Resorcinol and D) Phloroglucinol, with the column being 
polyethyleneimine-coated LiChrosorb Si 100 (10-micron particle diameter), 
25.times.0.4 cm.; the sample being 100 .mu.l containing 0.1 mg each 
compound; the eluent being 0.05 M potassium phosphate +0.1 M sodium 
acetate, pH 5.5, containing 10% methanol; the flow rate being 1.0 ml/min; 
the inlet pressure being 950 psi and the detection being A.sub.280 
monitored. 
Mononucleotides were resolved with a gradient, as shown in FIG. 6, for 
which a short (6.2 cm) column sufficed. In FIG. 6, 5'-Mononucleotides are 
shown as follows: A) CMP; B) AMP; C) UMP and D) GMP, with the column being 
polyethyleneimine-coated LiChrosorb Si 100 (10-micron particle diameter), 
6.2.times.0.4 cm; the sample being 100 .mu.l containing 0.07 mg each 
compound; the eluent being 3 min linear gradient (1.75 min delay); 0.01 M 
potassium phosphate, pH 3.0, to 1.0 M potassium phosphate, pH 2.0; the 
flow rate being 3.0 ml/ min; the inlet pressure being 250 psi and the 
detection being A.sub.254 monitored. 
EXAMPLE 2 
Demonstration of the Uniform Adsorption of Amines by Porous Silica 
1 to 2 grams of a porous silica (as specified hereinafter) were swirled and 
degassed in a 10% solution of polyethyleneimine 6 in methanol, then 
filtered and air-dried on a reduced-pressure funnel. 100 milligrams of the 
resulting coated, non-crosslinked material was removed for determination 
of the picric acid IEC. The remaining material was wetted and briefly 
slurried in the funnel with a stream of methanol, and again air-dried. 
This washing process was repeated eight times; after each wash, a sample 
of the material was removed for the picric acid assay. The graph of FIG. 7 
shows that adsorbed amine is readily washed down to a tightly-retained 
layer, presumably a monolayer, which is reached after 4 or 5 washes. 
Subsequent washes remove this layer much more slowly. Commerical HPLC 
silicas vary by as much as a factor of 4 both in the amount of amine 
initially adsorbed and in the amount retained in the monolayer. This 
demonstrates that the adsorption is an active process, and is affected by 
the composition of the silica surface. 
LiChrospher Si 500 (10-micron particle diameter) was coated with varying 
amounts of polyethyleneimine 6. The coated samples were crosslinked with a 
10% solution of pentaerythritol tetraglycidyl ether in dioxane as 
described in Example I and were then assayed for their picric acid and 
hemoglobin IEC. The graph of FIG. 8 shows that the hemoglobin IEC 
increases rapidly with the amount of surface amine (as determined with 
picric acid) until the amine concentration reaches 450 micromoles per gram 
of coated support. As the amount of amine increases beyond that 
concentration, the hemoglobin IEC rises much more slowly. This indicates 
that a monolayer coating of polyethyleneimine 6 on this particular silica 
contains 450 micromoles of amine residues per gram of coated support. The 
same value was obtained independently in the methanol washing experiment 
shown in FIG. 7. These results also suggest that the silica surface is 
completely covered by polyethyleneimine 6 at that concentration; the 
coating leaves no large bare patches. 
EXAMPLE 3 
Preparation of a Pellicular Coating on Porous Silica With Various Amines 
LiChrosorb Si 100 (10-micron particle diameter) was swirled and degassed in 
10% solutions of various amines (as specified hereinafter); the products 
were collected by filtration and crosslinked with pentaerythritol 
tetraglycidyl ether as described in Example 1. The amine contents of the 
resulting coatings were measured with the picric acid assay, as shown in 
Table 1, as follows: 
Table 1. 
______________________________________ 
Crosslinked amine coatings on 
LiChrosorb Si 100 (10-micron 
particle diameter) 
Picric Acid IEC, micromoles per 
Adsorbed Amine gram coated support 
______________________________________ 
N,N-Diethylethylenediamine 
310 
Ethylenediamine 1450 
Tetraethylenepentamine 
1470 
1,3-Diamino-2-hydroxypropane 
1530 
Polyethyleneimine 6 
1900 
______________________________________ 
As shown, these amines proved to be similar to each other and also to that 
of a monolayer of polyethyleneimine 6 on the same support; 1500-1600 
micromoles of amine per gram of coated support (see FIG. 7). This suggests 
that any small amine in excess of a monolayer is washed off during the 
crosslinking process. It also suggests that there is a 1:1 relationship 
between the surface adsorbing sites and the number of amine residues in a 
monolayer, whether the amine is simple or polymeric. 
An exception to these results was noted with N,N-Diethylethylenediamine 
which gave poor coating. This is due to the low number of active hydrogens 
in the molecule, which precluded extensive crosslinking. The resulting 
coating was unstable and washed off during the postcrosslinking treatment. 
EXAMPLE 4 
Use of Various Crosslinkers to Stabilize Pellicular Coatings on Porous 
Silica 
Two grams of LiChrospher Si 500 (10-micron particle diameter) were swirled 
and degassed in a 15% solution of polyethyleneimine 6 in methanol, then 
filtered out and air-dried on a reduced-pressure funnel. The coated, 
non-crosslinked product was divided into eight 270-milligram samples which 
were each crosslinked by a different agent as follows: 
Sample A: Swirled and degassed in 5 ml of dioxane containing 2 millimoles 
of 2-methyl-2-nitro-1,3-propanediol. The mixture was left three days at 
room temperature, then heated 30 minutes on a steam bath with occasional 
swirling. Product was collected by filtration and washed with acetone, 
water, diethylamine, water, and again acetone, then air-dried 
Sample B: Swirled and degassed in 5 ml of dioxane containing 2 millimoles 
of 1,3-dibromopropane. The mixture was left 24 hours at room temperature, 
then heated 30 min on a steambath with occasional swirling. The product 
was then filtered and washed as was Sample A. 
Sample C: Swirled and degassed in 5 ml of dioxane containing 2 millimoles 
of dithiobis (succinimidyl propionate). The mixture was left 24 hours at 
room temperature, then filtered and washed as was Sample A. 
Sample D: Swirled and degassed in 5 ml of dioxane containing 2 millimoles 
of ethylene glycol diglycidyl ether. The mixture was left 24 hours at room 
temperature, then heated 40 min on a steambath with occasional swirling. 
The product was filtered and washed as was Sample A. 
Sample E: Swirled and degassed in 5 ml of dioxane containing 2 millimoles 
of cyanuric chloride. The mixture was left 24 hours at room temperature, 
then filtered out and washed with methanol, water, diethylamine, water, 
methanol, and acetone, then air-dried. 
Sample F: Swirled and degassed in 5 ml of 0.01 molar sodium borate buffer 
(ph=9.2) containing 5 millimoles of dimethyl adipimidate dihydrochloride. 
The mixture was left 24 hours at room temperature, then filtered. The 
product was washed with water, diethylamine, water, and acetone, then 
air-dried. 
Sample G: Swirled and degassed in 5 ml of dioxane containing 2 millimoles 
of epichlorohydrin. The mixture was left 1.5 days at room temperature, 
then heated 40 min on a steambath with occasional swirling. The product 
was filtered and washed as was Sample A. 
Sample H (Control): Swirled and degassed in 5 ml of dioxane containing no 
crosslinker, heated 30 min on a steambath with occasional swirling, 
filtered, and washed as was Sample A. The product was air-dried. 
The products were assayed for their hemoglobin and picric acid IEC, as 
shown in Table 2 as follows: 
Table 2. 
______________________________________ 
IEC of Polyethyleneimine 6 Coatings on 
LiChrospher Si 500 (10-micron 
particle diameter) Crosslinked 
with Various Agents 
IEC per g Coated Support 
Picric Acid, 
Hemoglobin, 
Sample 
Crosslinker Micromoles Milligrams 
______________________________________ 
A 2-Methyl-2-nitro-1, 
3-propanediol 1030 61 
B 1,3-Dibromopropane 
1850 58 
C Dithiobis (succini- 
midyl propionate) 
620 30 
D Ethylene glycol di- 
glycidyl ether 1580 55 
E Cyanuric chloride 
362 26 
F Dimethyl adipimidate 
dihydrochloride 431 79 
G Epichlorohydrin 860 52 
H None 16 16 
______________________________________ 
Table 2 shows that the diepoxy resin, the nitro alcohol, and the alkyl 
bromide crosslinkers produced good anion-exchange coatings, both for small 
molecules and for proteins. The other agents were less suitable for the 
purpose. Dimethyl adipimidate dihydrochloride was an anomalous case, 
yielding a coating with a low small molecule IEC but a high protein IEC. 
In a similar experiment, a variety of epoxy resins were compared as 
crosslinking agents. 1.2 grams of LiChrosorb Si 100 (30-micron particle 
diameter) were coated with polyethyleneimine 6 from a 5% solution in 
methanol. 130-milligram samples of the product were then swirled and 
degassed in 5 of 10% (w/v) solutions of epoxy resins in dioxane. The 
mixtures were left 16 hours at room temperature, then heated 45 min on a 
steambath, with occasional swirling. The products were collected by 
filtration and washed with acetone, water, and again acetone, then 
air-dried. The IEC of the products were determined with the picric acid 
assay as shown in Table 3 as follows: 
Table 3. 
______________________________________ 
IEC of Polyethyleneimine 6 Coatings on 
LiChrosorb Si 500 (30-micron 
particle diameter) Crosslinked 
with Various Epoxy Resins 
Picric Acid IEC, Micromoles 
Epoxy Resin Crosslinker 
per g Coated Suport 
______________________________________ 
Pentaerythritol tetra- 
glycidyl ether 1420 
Epon 826 (Bisphenol A 
diglycidyl ether) 
1420 
Glyceryl diglycidyl ether 
1470 
Ethylene glycol diglycidyl 
ether 1510 
Epon 812 1470 
______________________________________ 
None of the coatings varied significantly in small molecule IEC. This 
indicates that any polyfunctional epoxy resin will produce a good, 
crosslinked anion-exchange coating with polyethyleneimine 6. The coating 
produced with Epon 826 would not be suitable for protein chromatography, 
since it contains aromatic residues; however, it may be useful for certain 
specialized applications. 
The crosslinkers described in Tables 2 and 3 are characterized in terms of 
the general structure (heretofore set forth) in Table 4 as follows: 
Table 4 
______________________________________ 
Structural Characteristics of Various 
Crosslinkers. 
Crossslinker (Type) 
A B C D 
______________________________________ 
1. 2-Methyl-2-nitro-1,3-propanediol (A=B.noteq.C.noteq.D) 
OH OH NO.sub.2 CH.sub.3 
2. 1,3-Dibromopropane (A=B.noteq.C=D) 
Br Br H H 
3. Dithiobis (succinimidyl propionate) (A=B) 
##STR5## 
##STR6## -- -- 
4. Ethylene glycol diglycidyl ether (A=B) 
glycidyl glycidyl -- -- 
5. Cyanuric chloride (A=B=C) 
Cl Cl Cl -- 
6. Dimethyl adipimidate dihydrochloride (A=B) 
##STR7## 
##STR8## -- -- 
7. Epichlorohydrin (A.noteq.B.noteq.C=D) 
##STR9## Cl H H 
8. Pentaerythrityl tetraglycidyl ether (A=B=C=C) 
glycidyl glycidyl glycidyl glycidyl 
9. Epon 826 (A=B.noteq.C=D) 
glycidyl glycidyl CH.sub.3 CH.sub.3 
10. Glyceryl diglycidyl ether (A=B.noteq.C.noteq.D) 
glycidyl glycidyl OH H 
11. Epon 812 (A=B) 
glycidyl glycidyl Not Well 
Characterized 
______________________________________ 
EXAMPLE 5 
Preparation of a Pellicular Polyethyleneimine Coating on Various Inorganic 
Materials 
200 milligrams of either silica, alumina or titania (see below) were 
swirled and degassed in a 10% solution of polyethyleneimine 6 in methanol. 
The materials were collected by filtration and air-dried on a 
reduced-pressure funnel. The coated supports were swirled and crosslinked 
in 10% solutions of pentaerythritol tetraglycidyl ether in dioxane, 11 
hours at room temperature and then 50 minutes on a steambath with 
occasional swirling. The products were then filtered out, washed with 
acetone, water, and again acetone, then air-dried. The small molecule IEC 
of the resulting coatings were determined with picric acid as shown in 
Table 5 below. A sample of controlled-pore glass was treated similarly, 
except that the crosslinking was effected by a 10% solution of Epon 812. 
Table 5 is as follows: 
Table 5. 
______________________________________ 
IEC of Crosslinked Polyethyleneimine 6 
Coatings on Various Inorganic 
Supports 
Picric Acid IEC, micromoles 
Materials per g Coated Support 
______________________________________ 
LiChrosorb Si 100 (10- 
micron particle diameter) 
1980 
Controlled-pore glass (5-10- 
micron particle diameter; 
100 A pore diameter) 
1600 
Spherisorb alumina (10- 
micron particle diameter; 
150 A pore diameter) 
780 
Bio-Rad basic alumina, 
Activity I (40-micron 
particle diameter) 
660 
Bio-Rad acid alumina, 
Activity I (40-micron 
particle diameter) 
620 
Corning titania (40/60 
mesh; pore diameter = 
400 A) 290 
______________________________________ 
Since alumina and titania are denser than silica, then comparing their 
coatings on the basis of support mass rather than volume leads to an 
underestimation of the performance of a column packed with coated alumina 
or titania. Such columns may be useful for applications at high pH, where 
silica is not stable. 
The pellicular anion-exchange coating (as described in Example 1, for 
example) has the following advantages over silane-based, covalently-bonded 
anion exchange coatings: 
(a) Ion-exchange capacity: The IEC of a supppot with a pellicular coating 
is up to 20 times greater than that of a support with a coating of an 
amine-containing silane. This permits resolution of some compounds, such 
as carboxylic acids, without a gradient. It also affords more efficient 
separations of substances such as proteins and nucleotides which are 
resolved with gradients. 
(b) Reproducibility: The IEC of different batches of HPLC supports with 
pellicular coatings differs by less than 10%. 
(c) Durability: Silane-based anion-exchange coatings are reported to 
degrade when eluted with aqueous buffers. No such degradation is observed 
with pellicular coatings, which are as stable as the underlying silica in 
aqueous media. Columns packed with pellicular materials last significantly 
longer than those with silane-based materials before the column fails, 
indicating that the pellicular coating also protects the underlying silica 
to some extent. 
Thus, this invention provides a high-capacity ion-exchange coating which 
can be applied to all inorganic materials used in high-performance liquid 
chromatography, whether porous or non-porous. 
The use of various amines, crosslinkers or inorganic materials is described 
in the examples set forth hereinabove and adsorption of the pellicular 
layer through an alternative force to ionic attraction is also described. 
In the examples given, the adsorption of the amine and its crosslinking on 
the surface of the support were performed with two different solutions. It 
is possible, however, to prepare a pellicular coating with a single 
solution containing both amine and crosslinker. One such procedure is to 
swirl and degas a support in a solution containing polyethyleneimine 6 and 
a polyfunctional epoxy resin. The material is collected by filtration and 
heated for one hour in an oven at 80.degree.. The product has a picric 
acid IEC equal to that of a material where the crosslinking was effected 
in a second solution. However, this method of preparation is not 
considered preferred since it could give less reproducible results and 
could tend to cement the support particles together. 
While the invention is well suited for use in liquid chromatography, 
materials with a pellicular amine coating could also be used in the field 
of industrial water processing. For example, inexpensive silicas with such 
a coating could be used to chelate metal ions and remove them from 
solution through simple filtration, and strongly anionic wastes could also 
be removed by such a procedure. In addition, immobilization of enzymes of 
industrial interest through adsorption could be carried out with the same 
inexpensive silicas. Such enzymes would retain their activity, and could 
be recovered through simple filtration of the mixture after their use as 
catalysts. 
As can be appreciated from the foregoing, this invention provides an 
improved process for producing a pellicular coating on a support material 
as well as an improved support with a thin coating adsorbed to and 
crosslinked on the support material.