U.S. patent number 3,722,181 [Application Number 05/039,665] was granted by the patent office on 1973-03-27 for chromatographic packing with chemically bonded organic stationary phases.
This patent grant is currently assigned to E. I. du Pont de Nemours and Company. Invention is credited to Joseph J. Kirkland, Paul C. Yates.
United States Patent |
3,722,181 |
Kirkland , et al. |
March 27, 1973 |
CHROMATOGRAPHIC PACKING WITH CHEMICALLY BONDED ORGANIC STATIONARY
PHASES
Abstract
A process for making a chromatographic packing having a
polymeric stationary phase in which molecules having the formula
wherein R.sub.3 ' is a hydroxyl, or an aliphatic or aromatic
hydrocarbon monovalent radical, and R.sub.4 is a monovalent
aliphatic or aromatic hydrocarbon radical, Are partially
prepolymerized, chemically bonded to a polyvalent metal-containing
substrate, the metal having a valence of 3-5, and further
polymerized. The polymeric stationary phase has a repeating unit of
the formula wherein A is --O-- or a monovalent aliphatic or
aromatic hydrocarbon radical, And is chemically bonded to the
surface of the substrate by an where silicon is part of a repeating
unit.
Inventors: |
Kirkland; Joseph J.
(Wilmington, DE), Yates; Paul C. (Wilmington, DE) |
Assignee: |
E. I. du Pont de Nemours and
Company (Wilmington, DE)
|
Family
ID: |
21906710 |
Appl.
No.: |
05/039,665 |
Filed: |
May 22, 1970 |
Current U.S.
Class: |
95/88; 96/101;
210/198.2; 210/656; 502/401 |
Current CPC
Class: |
B01J
20/3219 (20130101); C08G 65/22 (20130101); B01J
20/3276 (20130101); B01J 20/3272 (20130101); B01J
20/3257 (20130101); B01J 20/3204 (20130101); B01J
20/283 (20130101); B01J 20/3261 (20130101); C08G
83/001 (20130101); B01J 20/286 (20130101) |
Current International
Class: |
B01J
20/32 (20060101); B01J 20/30 (20060101); C08G
65/00 (20060101); C08G 65/22 (20060101); C08G
83/00 (20060101); B01d 015/08 () |
Field of
Search: |
;252/431 ;55/67,197,386
;73/23.1 ;210/31,198 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Adee; John
Claims
What is claimed is:
1. An apparatus for use in chromatographic separations comprising a
resolving zone through which materials to be separated and a
carrier fluid are passed, said resolving zone comprising a packing
having a polyvalent metal-containing substrate and a stationary
phase, said stationary phase comprising a polymer being
sufficiently porous to allow penetration by said materials and
carrier fluid and having the repeating unit of the formula
wherein A is --O-- or a monovalent aliphatic or aromatic
hydrocarbon radical and R is a monovalent aliphatic or aromatic
hydrocarbon radical,
said metal of said substrate having a valence of 3-5, said
stationary phase being chemically bonded to the surface of said
substrate by an
linkage, wherein M is said polyvalent metal and
is part of one of said repeating units of said stationary
phase.
2. An improved process for performing chromatographic separations
comprising:
a. placing the material to be separated in a carrier fluid;
b. contacting said material and said carrier fluid with a packing
having a polyvalent metal-containing substrate and a stationary
phase, said stationary phase comprising a polymer being
sufficiently porous to allow penetration by said material and
carrier fluid and having the repeating unit of the formula
wherein A is --O-- or a monovalent aliphatic or aromatic
hydrocarbon radical and R is a monovalent or aliphatic or aromatic
hydrocarbon radical,
said metal of said substrate having a valence of 3-5, said
stationary phase being chemically bonded to the surface of said
substrate by an
linkage, wherein M is said polyvalent metal and
is part of one of said repeating units of said stationary
phase.
3. An apparatus for use in chromatographic separations comprising a
resolving zone through which materials to be separated are passed,
said resolving zone comprising a packing having a polyvalent
metal-containing substrate and a stationary phase having an average
thickness of about 30 A.degree. to 10,000 A.degree., said
stationary phase comprising a porous polymer having the repeating
unit of the formula
wherein A is --O-- or a monovalent aliphatic or aromatic
hydrocarbon radical and R is a monovalent aliphatic or aromatic
hydrocarbon radical,
said metal of said substrate having a valence of 3-5, said
stationary phase being chemically bonded to the surface of said
substrate by an
linkage, wherein M is said polyvalent metal and
is part of one of said repeating units of said stationary phase,
and said polymer comprising 5 to 80 percent of the volume of said
stationary phase.
4. The apparatus of claim 3 wherein said metal is silicon.
5. The apparatus of claim 3 wherein A is --O--.
6. The apparatus of claim 3 wherein said A is a monovalent
aliphatic or aromatic hydrocarbon radical.
7. The apparatus of claim 3 wherein said stationary phase comprises
at least two copolymerized portions, A in the first of said
portions being --O--, and said A in the second of said portions
being an aliphatic or aromatic hydrocarbon.
8. The apparatus of claim 3 wherein said packing is different in
different parts of said resolving zone.
9. The apparatus of claim 3 wherein said resolving zone is disposed
within a column.
10. The apparatus of claim 3 wherein said resolving zone comprises
a thin layer upon a surface.
11. An improved process for performing chromatographic separations
comprising:
a. placing the material to be separated in a carrier fluid;
b. contacting said material and said carrier fluid with a packing
having a polyvalent metal-containing substrate and a stationary
phase having an average thickness of about from 30 A.degree. to
10,000 A.degree., said stationary phase comprising a porous polymer
having the repeating unit of the formula
wherein A is --O-- or a monovalent aliphatic or aromatic
hydrocarbon radical and R is a monovalent or aliphatic or aromatic
hydrocarbon radical,
said metal of said substrate having a valence of 3-5, said
stationary phase being chemically bonded to the surface of said
substrate by an
linkage, wherein M is said polyvalent metal and
is part of one of said repeating units of said stationary phase,
and said polymer comprising about 5 to 80 percent of the volume of
said stationary phase.
12. The process of claim 11 wherein said metal is silicon.
13. The process of claim 11 wherein A is --O--.
14. The process of claim 11 wherein A is a monovalent aliphatic or
aromatic hydrocarbon radical.
15. The process of claim 11 wherein said packing comprises at least
two copolymerized portions, A in the first of said portions being
--O--, and A in the second of said portions being a monovalent
aliphatic or aromatic hydrocarbon radical.
16. The process of claim 11 wherein said packing is different in
different parts of said column.
17. The process of claim 11 wherein said resolving zone is disposed
within a column.
18. The process of claim 11 wherein packing comprises a thin layer
upon a surface.
Description
BACKGROUND OF THE INVENTION
This invention relates to improved chromatographic packings in
which organosilanes are chemically bonded to a substrate and
polymerized to form a stationary organic phase.
A previous attempt to chemically bond a stationary phase to a
substrate involved the esterification of siliceous chromatographic
supports with alcohols. However, the resulting Si -- O -- C linkage
was hydrolytically unstable and the product consisted of
monomolecular films that only allow adsorptive, but not partition,
interactions with the solute.
Further, polymolecular silicones have been reacted to
chromatographic supports by employing two series of steps. In the
first series, dimethyldichlorosilane or methyltrichlorosilane is
bonded to the silica substrate. In the second series,
organochlorosilanes are attached to the above methylchlorosilanes.
Due to the use of highly reactive chlorosilanes in both series of
steps, the extent of reaction in each series is difficult to
control, which usually results in films of variable thickness. In
addition, the first series of steps places methyl groups on the
substrate surface which reduces the effective polarity of the
packing. This clearly represents a detriment when polarity is
desired. Additionally, the second series of steps does not permit
the use of volatile organochlorosiles, thus restricting the choice
of resultant stationary phases. Further, the particular
organochlorosilanes that would be required as starting reagents to
place certain functional (e.g., amino) groups on the surface would
be self-reactive and thus self-destructive. Lastly, it was
necessary,to deactivate the remaining active surface sites before
chromatographic use. Deactivation leaves a silicate ester which has
sufficient thermal and hydrolytic unstability to cause "bleed" at
the higher temperatures used in gas chromatography.
Chemical bonding of silanes as "coupling agents" to
silica-containing surfaces is known in the prior art where the
surface is contacted with an aqueous solution of the coupling
agent, and the water removed. The resulting bonded layer lacks
porosity, has uncontrollable thickness, and hence, is unsuitable
for chromatography, which must have both to allow for precise
diffusion of the substances being chromatographed.
Thus it is an object of this invention to provide a chromatographic
packing with an organic stationary phase chemically bonded directly
to a silica-containing substrate with a bond that is hydrolytically
and thermally stable.
It is a further object to provide a process for producing such a
packing wherein the thickness and porosity of the resulting
chemically-bonded stationary phase are controllable.
SUMMARY OF THE INVENTION
These and other objects are accomplished by prepolymerizing
hydroxysilane reagents in the presence of a strictly limited
quantity of water. After prepolymerization, the silane reagent is
reacted to the surface of the substrate, and further polymerized to
form the chemically-bonded stationary phase. The silane reagent
molecules have the general formula
wherein R.sub.3 ' is a hydroxyl or a monovalent aliphatic or
aromatic hydrocarbon radical, and
R.sub.4 is a monovalent aliphatic or aromatic hydrocarbon. R.sub.3
' or R.sub.4 may contain atoms other than carbon and hydrogen. The
bonded polymolecular stationary phase has a repeating unit with the
formula
wherein A is a monovalent aliphatic or aromatic hydrocarbon
radical. The stationary phase is bonded to the polyvalent
metal-containing substrate surface through an
linkage, where M is the metal and
is part of a repeating unit.
By choice of R.sub.4 and (when not a hydroxyl) R.sub.3 ', the
chemically bonded stationary phase may be produced with a variety
of functional groups, resulting in chromatographic packing with
widely diverse selectivity. The resulting packings may vary from
extremely polar to non-polar according to the needs of the
particular separation to be performed.
The thickness of the resulting stationary phase is controlled by
controlling the quantities of starting materials. Thus, bonded
stationary phase may be deposited that is controllably
monomolecularly or polymolecularly thick. In particular, a
polymolecular layer of predetermined thickness, which will allow
for precise diffusions, may be bonded to the substrate.
Mixtures of the above starting compounds may be reacted to the
surface and copolymerized to give "blends" for particular purposes.
The compositions of these blends are determined by controlling the
concentrations of initial reactants.
A particularly useful type of blend is one by which the degree of
cross-linking in the bonded and polymerized stationary phase can be
controlled. Greater concentrations of starting reagents in which
R.sub.3 ' is a hydroxyl result in higher degree of cross-linking,
which is particularly useful for gas chromatography (hereinafter
referred to as G.C.). On the other hand, greater concentrations of
compounds with R.sub.3 ' being non-hydroxyl result in less
cross-linking which is desired for liquid chromatography
(hereinafter referred to as L.C.). The extreme degrees of
cross-linking are obtained by using either one of these (hydroxyl
or non-hydroxyl) alone.
The use in L.C. of packings with the chemically bonded stationary
phase eliminates the necessity for precolumns or presaturating the
carrier with the stationary phase. Further, high column
efficiencies are maintained because of the homogeneous distribution
of the stationary phase on the surface of the supports. This bonded
stationary phase provides greater column stability and eliminates
many problems associated with the loss of partitioning liquid
during the operation of conventional liquid - liquid
chromatographic columns.
G.C. substrates with the chemically bound liquid stationary phase
show very low vapor pressure. Thus column life is extended and the
level of "noise" in the detection system due to stationary phase
"bleed" is minimal. These packings also show very high thermal
stability as preferred for G.C.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a diagrammatical representation of a cross-section of a
preferred chromatographic support material.
FIG. 2 is a schematic cross-section of a polymeric stationary phase
chemically bonded to a substrate.
FIG. 3 shows the chemical structure of the polymeric stationary
phase.
FIG. 4 is a part of the liquid chromatographic separation of
sulfonamides using the present invention.
FIG. 5 compares liquid chromatographic HETP v. Carrier Velocity
Plots of the present invention with the prior art.
FIG. 6 is a plot of the liquid chromatographic separation of
thiolhydroxamates using the present invention.
FIG. 7 is a plot of the liquid chromatographic separation with a
"nitrile" bonded phase column of the present invention.
FIG. 8 is a plot showing a programmed temperature gas
chromatographic separation in an "ether" bonded-phase of the
present invention.
FIG. 9 compares chromatograms showing the selectivity in gas
chromatography of the "ether" bonded-phase of the present
invention.
FIG. 10 is a plot showing the relative "bleed" rate of a "nitrile"
stationary bonded-phase of the present invention as against a
column of the prior art.
FIG. 11 is a plot of a thermograviometric curve for a chemically
bonded "nitrile" packing of the present invention.
FIG. 12 is a plot of gas chromatographic separation of aromatic
hydrocarbons with a "nitrile" bonded-phase.
DETAILED DESCRIPTION OF THE INVENTION
Chromatographic packings may be prepared having chemically bonded
organic stationary phases with a variety of functional groups,
resulting in widely diverse chromatographic selectivity.
Bonded-phase packings may be prepared for gas and liquid
chromatography on a variety of substrates. The composition of the
substrate is not critical, except that its surface must be capable
of chemically reacting with silanols. Silicacontaining substrates
are preferred, although any polyvalent metal-containing substrate
may be used where the valence of the metal is from three through
five. Examples of useful substrates include diatomaceous earths,
silica gel, glasses, sand, aluminosilicates, quartz, porous silica
beads, and clays. Additionally, non-alkaline metal oxides, alumina,
thoria, titania, zirconia, and non-alkaline metals with an oxide
skin may be utilized.
When the substrate is in the form of a support (i.e., particulate),
the shape of the particles is not critical. Examples of shapes
include rings, polyhedra, saddles, platelets, fibers, hollow tubes,
rods, or cylinders. Spherical supports are preferred because of
their regular and reproducible packing characteristics and ease and
convenience in handling.
The controlled surface porosity, support of FIG. 1 (described in
U.S. Pat. No. 3,505,785 and sold under the trade name "Zipax" by E.
I. du Pont de Nemours and Company, Wilmington, Del.) is a preferred
embodiment of a support structure. It has an impervious core 11,
preferably of glass or other ceramic material and a porous coating
15 made up of sequentially adsorbed monolayers 13 of
microparticles. It consists of discrete spherical particles having
a large number of superficial shallow pores 12 and no deep pores.
It has regular and reproducible packing characteristics, ease and
convenience in handling, and desirable characteristics for
high-speed, high-efficiency gas and liquid chromatography.
In discussing substrates, the terms "surface," "surface area ",
"available surface," etc. refer to all surfaces which are
accessible from the exterior of the substrate. In FIG. 1, for
example, the surfaces within the pores 12 would be included in
these terms.
It is often desirable for the support or substrate surface to have
a high initial population of metal-hydroxy, or preferably --SiOH
groups. This is best accomplished by treating the surface with acid
or base. The preferred procedure is to heat the support with
concentrated nitric or hydrochloric acid on a steam bath for
several hours. Excess acid or base should be removed from the
surface by thorough washing before the support is dried for
reaction with the silane reagents.
The reagents employed to produce the chemically bonded organic
stationary phase are silanols having the formula
where R.sub.3 ' and R.sub.4 are given above. They are preferably
prepared from silicate esters with the formula
where R.sub.1 and R.sub.2 are alkoxy, R.sub.3 is alkoxy or R.sub.3
', and
R.sub.4 is given above.
A list of commercially available reagents is shown in Table I.
Table II gives other reagents which are not presently known to be
commercially available. Their structures encompass a variety of
configurations. These may be synthesized according to the general
techniques contained in Chemistry of the Silicones, by R. G.
Rochow, John Wiley & Sons, New York, 2nd Ed. 1951. ##SPC1##
##SPC2##
A silicate ester, when used as the reagent, must first be
hydrolyzed to the silanol which then engages in prepolymerization,
reaction with the substrate surface, and subsequent polymerization.
Factors which affect this hydrolysis include:
1. high or low pH (bases or acids),
2. presence of soluble salts of certain metals such as copper,
lead, zinc, and iron, and
3. temperature.
In general, the silane ester (except for aminosilanes), is
hydrolyzed under acidic conditions preferably at elevated
temperatures. For the subsequent prepolymerization, the hydrolysis
is preferably carried out by refluxing the reagent in a non-aqueous
solvent containing several molar excesses of water with an acid
catalyst. The solvent in addition to having some solubility for
water should be non-reactive with the reagent water or the (acid or
base) catalyst. It should boil in the range of about 25.degree. to
300.degree.C., preferably 50.degree. to 200.degree.C. Ethers such
as tetrahydrofuran and dioxane have worked satisfactorily.
The amount of water added to the non-aqueous solvent for the
hydrolysis should be at least one-third of the equivalent of the
silane reagent and need not be more than about a 100-fold
equivalent excess. One equivalent to a 50-fold equivalent excess
generally suffices. A very large excess of water inhibits the
subsequent partial polymerization. The hydrolysis is normally run
at elevated temperatures (preferably the boiling point of the
non-aqueous solvent) for a period of about 30 minutes to several
hours.
The operation is generally performed longer than required for
hydrolysis, in order to effect partial polymerization. If necessary
for the partial polymerization, the water content and pH of the
solution may be adjusted, the latter to a value of from 3 through 6
by ammonia, acetic acid, or some other catalyst.
Partial polymerization of the silanes before reaction to the
substrate is essential in producing a chemically bonded stationary
phase having the required thickness and porosity. Initiation of
polymerization may be determined by placing a small amount of the
silane-containing solution in water. The appearance of cloudiness
indicates that the prepolymerization, having progressed to the
point where at least part of the silane reagent is insoluble in
water, is sufficient. Contrariwise, this prepolymerization should
not progress so far as to appreciably increase the viscosity of the
silane-containing solution.
The reaction of the prepolymerized silane to the substrate,
followed by the final polymerization, is carried out at
temperatures from about 50.degree. to 350.degree.C., a range of
100.degree.-250.degree.C. being preferred. Times required for this
step vary from a few minutes to several hours, but usually 1-3
hours is adequate for the preferred temperature range.
Other procedures in which silane ester reagents may be
prepolymerized and reacted to the chromatographic substrates
include:
1. Preparing an acidic (pH 3 to 5) aqueous solution of the reagent
whereby it hydrolyzes to the corresponding silanol compound. A
suitable chromatographic substrate is soaked in the solution and
excess liquid filtered off. The mixture is first heated to a
temperature below 100.degree.C. to initiate polymerization, and
then further heated to carry out the reaction to the substrate
surface and to complete polymerization to form the polymeric
organic phase.
2. The silane ester reagent is deposited on the chromatographic
substrate by evaporation from a suitable volatile solvent. The
reagent is then hydrolyzed and prepolymerized by passing moist air
or steam through the coated support. The reaction with the surface
and polymerization is then carried out by heating.
3. The silicate ester reagent contained in a volatile unreactive
solvent such as tetrahydrofuran is hydrolyzed and prepolymerized by
adding aqueous hydrochloric acid and refluxing the mixture. The
substrate is added to this solution. The volatile organic solvent
is then removed by low temperature vacuum distillation. A large
excess of toluene, xylene or other suitable solvent is added to the
wet powder and the water-solvent azeotrope is continuously removed
until the reaction and polymerization is complete.
4. Chemical means may be used to remove the water which is the
driving force in these reactions. The addition of reagents that
readily react with water, such as dimethoxypropane, will effect the
desired prepolymerization, the reaction to the substrate surface,
and the subsequent polymerization.
A final extraction step may be used to remove any silane not bonded
to the substrate. The solvents used in this step should have some
solubility for the unbonded polymer which insures that the polymer
left on the substrate is indeed chemically bonded thereto.
Mixtures of different types of silane molecules may also be used in
the prepolymerization, reaction, and polymerization steps. In this
manner, bonded stationary phases having "blends" of various
properties suitable for particular separations may be
developed.
A particularly useful type of "blend" is the one controlling the
extent of cross-linking in the polymerization process. Packings for
gas chromatography generally require a highly cross-linked
structure, in order for the polymer to have optimum stability at
high temperatures. Conversely, packings for liquid chromatography
should have a less cross-linked polymeric phase. This lower order
of cross-linking permits better penetration of the carrier, with
subsequent improved accessibility of the solute into the solvated
polymeric structure. The chemically bonded stationary phases also
possess unique chemical and physical properties because of their
ability to swell in certain solvents and to form gelatinous,
ordered structures which function as selective stationary gel
phases.
The extent of the cross-linking reaction is controlled by adding
dialkoxysilicates,
to the usual trialkoxysilicate, R -- Si (OR').sub.3, reagents. The
addition of dialkoxysilicates to the reaction significantly reduces
the cross-linking, since the other group (R") attached to the
silicon atom does not engage in the polymerization. The addition of
the modifying functional group R" also may impart desirable
physical and chemical properties to the bonded phase.
As shown in the examples below, the reaction of the silicate
reagents to the substrate surface proceeds very close to
quantitatively. Thus, by controlling the initial amounts of
reagents, it is possible to determined the thickness of the
resulting film of chemically bonded stationary phase upon the
substrate surface. A monolayer film may be produced, for example,
where it is desired to effect selective adsorption. However, in the
usual partition chromatography, a substrate with a polymolecular
layer is utilized.
In the latter case the polymeric stationary phase is basically a
three-dimensional porous network of repetitive, functional groups
of the silane structure, chemically bonded to the support surface
by an
A schematic cross-section representation of the porous polymer
structure is shown in FIG. 2 where the substrate is indicated at
20, the chemically bonded stationary phase at 21, a portion of
polymer at 22, and the pores at 23. FIG. 3 gives an exploded
schematic representation of the claimed structure of a piece of
polymer 22.
This chemically-bonded organic phase should have an average
thickness of 30 A.degree. to 10,000 A.degree., with
50.degree.-2,000 A.degree. being preferred. This preferred range
encompasses sufficient thickness to ensure the desired
chromatographic interactions without being so large (thick) so as
to greatly affect column efficiency as a result of resistance to
mass transfer of the solute within the polymeric phase. The bonded
stationary phase should have about 20 to 95 percent of its volume
consisting of pores, with a range of 35 to 75 percent preferred.
The polymer should be sufficiently porous to allow penetration by
the carrier phase and the sample components, while having
sufficient cross-linking to ensure that the desired mechanical and
chemical stability will be obtained.
Usually in chromatography, the substrate is a particulate support.
These particles are coated with the stationary phase and packed
into columns. In the present invention, this stationary phase is
chemically bonded to the support particles, which are then packed
into columns in the usual way. However, the present invention is
also useful in other forms of chromatography. The stationary phase
may be bonded to a substrate of silica gel. Also, where a capillary
tube without a particulate support is used for the column, a
stationary phase may be bonded directly onto the column wall
itself.
The bonded-phase materials are also useful for "liquid-partition"
thin layer chromatography. The procedure is to coat a suitable thin
layer adsorbent, such as finely divided (5 to 10 microns) silica
gel or diatomaceous earth with one of the bonded polymers. This
chromatographically active material is coated in thin layers on
plates in the usual manner. The plates are usually glass and flat,
but other materials and shapes may be used. Mixtures of compounds
are then spotted on these thin layer plates and developed, using
the usual procedures for liquid-adsorption thin layer
chromatography. This approach permits the thin layer chromatography
of relatively insoluble compounds to be carried out with polar
solvents in a liquid-liquid partitioning mode. The techniques of
liquid-liquid thin layer chromatography are generally difficult,
and chromatographic separations of sparingly soluble compounds by
the liquid-liquid partition approach is generally impossible
without the use of a chemically bound stationary phase.
In liquid chromatography, use of packings with bonded stationary
phases provides additional operational advantages over columns with
conventional mechanically held liquid phases. Peaks may be
collected and the constituent readily isolated for further
characterization by simply evaporating the volatile carrier system.
This is possible because the sample is not contaminated by a less
volatile stationary phase which is usually present in
carrier-saturation amounts in a conventional liquid-liquid system.
Further, it is practical to recycle the effluent from the detector
back into the reservoir for further use, without any treatment,
since the carrier need not be equilibrated with a stationary
phase.
Packings with chemically-bound stationary phases are ideal for
gradient elution and flow-programmed liquid chromatographic
separations. The polarity and flowrate of the carrier solvent may
be changed stepwise or continuously during a run without degrading
the column. Thus, it is possible to chromatograph a sample having
components with widely varying partition ratios in a single
chromatographic run in a manner that assures that all of the sample
constituents are eluted. "Regeneration" of the bonded-phase packing
after a gradient elution run is accomplished rapidly.
EXAMPLE 1
Preparation of "Ether"/"Zipax" Packing for Liquid
Chromatography
Twenty-five grams of <40 microns "Zipax" and 150 ml. of
concentrated nitric acid are placed in a 250 ml. beaker and heated
on a steam bath for 2 hours with occasional stirring. The resulting
support is washed free of acid by repeatedly slurrying with
distilled water. The material is then air-dried on a Buchner
sintered glass vacuum filter and heated for 1 hour at 125.degree.C.
in a circulating air oven.
To a 100 ml. round bottom flask is added 25 ml. of fresh reagent
grade tetrahydrofuran (Fisher Scientific Co.), 6.65 ml. of a 50
mg./ml. tetrahydrofuran solution of Dow Corning Z-6040
(.gamma.-glycidoxypropyltrimethoxysilane) and 0.75 ml. of 0.01N
hydrochloric acid. This mixture is refluxed gently under a
condenser for 30 minutes. This hydrolysis mixture is then added to
the 25 grams of acid-treated "Zipax" from above, contained in a
shallow evaporating dish. The volatile solvent is removed while
stirring the mixture under a slow stream of nitrogen. The resulting
dry powder is then heated for 1 hour at 125.degree.C. in a
circulating air oven. When cooled, the mixture is then transferred
to a 500 ml. round bottom flask containing 150 ml. of dioxane. This
mixture is refluxed with gentle stirring for 30 minutes. The
solvent is decanted from the support and 150 ml. of reagent grade
methanol added to the flask. The resulting mixture is refluxed for
10 minutes. The solvent is then decanted, and another 150 ml. of
absolute methanol added and again the mixture refluxed for 10
minutes. The solvent is decanted and the resulting support air
dried on a sintered glass Buchner funnel. The support is then dried
in a circulating air oven for 30 minutes at 125.degree.C.
Elemental analysis of the resulting "ether" bonded-phase carried
out on an F & M Scientific Co. Model 185 C, H & N analyzer
showed 0.35, 0.33 percent carbon, and 0.062, 0,63 percent hydrogen.
Assuming the reaction depicted below, elemental data show that 0.77
percent polymer was present on the "Zipax," representing 88 percent
of theoretical. ##SPC3##
Scanning electron micrographs of this bonded-phase material show no
differences when compared with the starting uncoated "Zipax,"
indicating that the silane is reacted with the microparticle
surface and that the resulting stationary phase does not obstruct
the pores.
The porosity of the polymeric stationary phase itself is evidenced
by surface area measurements made on "Zipax" coated with the
following amounts of the "ether" bonded-phase.
Material Surf. Area, m.sup.2 /g. Uncoated "Zipax" 1.0 0.38 %
"ether" 0.11 1.16% "ether" 0.19 1.65% "ether" 0.42
The rapid decrease in surface area when "Zipax" is coated with a
small amount of polymer indicates that initially, the polymer
reacts in the pores between the microparticles. The surface area
then rises as the amount of polymer bonded to the support increases
clearly showing that the bonded stationary phase is indeed porous.
Also, the stationary phase is not extracted by boiling organic
solvents which would normally have significant solubility for the
polymer indicating that the polymer has actually reacted with the
"Zipax" surface.
An illustrative chromatographic separation using this
"ether"/"Zipax" boned-phase packing is shown in FIG. 4. This
mixture of aromatic sulfonamides was separated in about 6.5 minutes
with a 1 meter .times. 2.1 mm i.d., column, using a carrier of 5
percent chloroform in hexane at 27.degree.C., a flow of 2.66
cc./min., a column input pressure of 860 psi, and an ultraviolet
detector sensitivity of 0.05 absorbance, full-scale. The apparatus
employed was described in an article by Joseph J. Kirkland in
Journal of Chromatograph Science 7, 7 (1969).
The "ether" bonded-phase column used to separate these sulfonamides
is unaffected by the chloroform in the carrier. Had this separation
been carried out with a conventional system, the polar solvent
would have had appreciable solubility for the stationary liquid,
necessitating presaturation of the carrier by the stationary phase
and a pre-equilibrating column containing packing with the
stationary phase. None of these precautions were required with a
bonded-phase column.
The "ether" bonded-phase column demonstrates stability at high
carrier velocities. The column was operated at a carrier linear
velocity of 20.8 cm./sec. (column input pressure - 3,000 psi) with
no apparent change in chromatographic properties in succeeding use.
Conventional liquid-liquid chromatographic columns are maintained
with extreme difficulty because the relatively high shear forces
developed at these flow rates cause the loss of the stationary
liquid phase from the packing.
The efficiency of the dry-packed "ether" bonded-phase column is
nearly equivalent to that of comparable liquid-liquid
chromatographic columns made with mechanically held stationary
phases. FIG. 5 shows comparative HETP (height equivalent to a
theoretical plate) versus linear carrier velocity plots for two
columns made from the same original 325-400 mesh "Zipax" supports.
One column embodies mechanically-held 1 percent
.beta.,.beta.-oxydipropionitrile as the stationary phase, while the
other consists of a 0.94 percent "ether" bonded-phase. HETP data
for the bonded-phase column is slightly higher than that for the
conventional liquid-liquid column for two solutes of which
acetophenone is essentially unretarded and benzyl alcohol is
moderately retained.
After obtaining the original HETP data, the "ether" bonded-phase
column was operated at input pressures up to 5,000 psi and carrier
linear velocities up to 40 cm./sec. Even under these very drastic
conditions, the column showed little degradation, as evidenced by
the especially marked points 25 in the FIG. 5 plot. Operation of
conventional liquid-liquid chromatographic columns at carrier
velocities of 40 cm./sec. is very difficult, because of the loss of
mechanically held stationary phase.
The uniqueness of the "ether" bonded-phase for liquid
chromatographic separations is further illustrated in FIG. 6. This
synthetic mixture of the thiolhydroxamates shown was separated in a
1 meter .times. 2.1 mm. i.d., column of the 325-400 mesh "Zipax"
containing 0.94 percent "ether" bonded-phase, the column being
operated at 27.degree.C., with a carrier of 10 percent chloroform
in hexane. These compounds are difficult to separate with
conventional liquid-liquid chromatographic systems because of their
polyfunctionality and their high polarity. They are strongly
retained on most conventional stationary phases, and require a
polar carrier to elute them in a reasonable time. The solubility of
the stationary phase in this rapidly moving polar carrier causes
extreme difficulty in maintaining a column of constant performance.
No such difficulties are experienced with the "ether" bonded-phase
packing, and carriers having any desired polarity can be used to
chromatograph highly polar compounds.
EXAMPLE 2
Preparation of ".beta.-Cyanoethyl"/"Zypax"
Bonded-Phase
Twenty-five milliliters of reagent grade dioxane (Special Services,
Du Pont Experimental Station), 5.5 ml. of 70 mg./ml. General
Electric XC-3711 (.beta.-cyanoethyltriethoxysilane) in dioxane, and
2 ml. of 0.1N hydrochloric acid are combined in a 250 ml. round
bottom flask and refluxed for 1 hour under a condenser. The
resulting hydrolyzed mixture is poured onto 25 grams of <400
mesh "Zipax" (acid-washed as described above), and the solvent
removed while continuously stirring under warm air from a heat gun.
The resulting dry powder is heated for 1 hour at 150.degree.C. in a
circulating air oven. The treated material is transferred to a 500
ml. roundbottom flask and refluxed with 200 ml. of absolute
methanol for 15 minutes. The slightly cloudy solvent is decanted
and the support again refluxed with 200 ml. of fresh absolute
methanol for 15 minutes. The clear solvent is decanted and the
support again refluxed for 15 minutes with a fresh 200 ml. portion
of absolute methanol. The clear solvent is decanted, the treated
support air dried on a sintered glass Buchner filter funnel, and
then heated in a circulating air oven for 30 minutes at
150.degree.C.
By elemental analysis, the resulting bonded-phase showed 0.23,
0.232 percent carbon, 0.032, 0.034 percent hydrogen, and 0.087,
0.089 percent nitrogen. These data indicate that the packing
material contained 0.83 percent silicone polymer (calculated on the
structure proposed below), or 90 percent of theoretical. Nitrogen
absorption (flow method) showed that the surface area of this
material was 0.44 m.sup.2 /g. ##SPC4##
An example of the selectivity of the "nitrile" bonded-phase packing
for liquid chromatography is shown in FIG. 7. This synthetic
mixture of relatively insoluble substituted acetanilides and
sulfonamides was separated in about 5 minutes with a high flowrate
of 1:1 isopropylchloride-hexane. Use of this very polar carrier in
conventional liquid-liquid chromatography is questionable because
of its high mutual solubility with most liquid stationary phases.
It is noteworthy that this chemically held "nitrile" packing
selectively retains compounds which can readily hydrogen-bond.
Compounds with acidic --NH.sub.2 groups are particularly retarded,
and certain substituted phenolics, such as 4-acetamidophenol, are
also highly retained.
The .alpha.-cyanoacetanilide peak in FIG. 7 has a HETP of 1.89 mm.
at a flowrate of 4.35 cc./min., which corresponds to 8.0
theoretical plates/sec. (4.8 effective plates/sec.). When operated
at a carrier linear velocity of 1 cm./sec. or less, this column
demonstrates HETP of less than 1 mm. for similar solutes.
The efficiency of the bonded-phase liquid chromatographic columns
depends on the type and polarity of the carrier used. Columns with
"nitrile" bonded-phase packing show poor efficiency with hexane as
carrier. However, as the polarity of the carrier is increased, so
does the efficiency of the column (equal solute partition
ratios).
EXAMPLE 3
Preparation of "Ester"/"Zipax"Bonded-Phase
To a 100 ml. round-bottom flask is added 25 ml. of reagent grade
dioxane, 2 ml. of 0.1N hydrochloric acid, and 4 ml. of a 70 mg./ml.
solution of Union Carbide Silane A-174
(.gamma.-methacryloxy-propyltrimethoxysilane) in dioxane. The
resulting mixture is refluxed gently for 1 hour under a condenser.
This hydrolysis mixture is then added to 20 grams of <400 mesh
"Zipax" contained in a shallow evaporating dish, and the solvent
removed while gently stirring the mixture under warm air from a
heat gun. The resulting mixture is then heated for 1 hour at
150.degree.C. in a circulating air oven. The treated support is
then transferred to a 500 ml. round-bottom flask which contains 200
ml. of absolute methanol. The mixture is refluxed for 15 minutes,
the clear solvent decanted, and this extractive technique repeated
two more times with fresh 200 ml. portions of absolute methanol.
After the third extraction, the material is filtered off on a
sintered glass Buchner filter funnel and air-dried. The treated
support is then dried to 150.degree.C. for 30 minutes in a
circulating air oven.
Elemental analysis of the "ester" bonded-phase showed carbon 0.443
percent, hydrogen 0.077 percent, indicating that 1.07 percent (96
percent of theory by carbon analysis) of the polymer phase below
was present on the support. The surface area of this product by
nitrogen adsorption (flow method) was 0.52 m.sup.2 /g. ##SPC5##
EXAMPLE 4
Preparation of "Ether" Bonded-Phase
for Gas Chromatography
Fifty grams of 100-140 mesh "Zipax" (nitrogen surface area 0.46
m.sup.2 /g.) is heated in a muffle furnace at 725.degree.C. for 1
hour, then cooled in a desiccator. The resulting material is then
treated with nitric acid in the manner described in Example 1,
above.
To a 100 ml. round-bottom flask is added 20 ml. of reagent grade
dioxane, 10 ml. of 100 mg./ml. Dow Corning Z6040
(.gamma.-glycidoxypropyltrimethoxysilane) and 5 ml. of 0.1N
hydrochloric acid. The mixture is refluxed under a condenser for 1
hour and then added to the 100-140 mesh acid-treated "Zipax"
described above. The volatile solvent is removed from the solution
while stirring the mixture under warm air from a heat gun. The
resulting material is heated at 150.degree.C. for one hour in a
circulating air oven. The support is then placed in a 500 ml.
round-bottom flask with 200 ml. of absolute methanol and refluxed
with gentle agitation for 15 minutes. The solvent is decanted and
the extraction carried out twice more with fresh 200 ml. portions
of absolute methanol. The thrice-extracted material is filtered off
onto a sintered glass Buchner filter funnel, air-dried and then
heated in a circulating air oven at 150.degree.C. for 30 minutes.
Elemental analysis of the sample showed 0.50 percent carbon,
corresponding to 1.1 percent polymer on the "Zipax" surface, or 82
percent of theoretical.
Use of the "ether" bonded-phase packing for a gas chromatographic
separation is shown in FIG. 8. This separation was carried out on a
1 meter .times. 1/4 inch o.d., 1/8 inch i.d. glass column, using
helium carrier gas flowrate of 50 cc./min. and a flame ionization
detector sensitivity of 1 .times. 10.sup.-.sup.9 amp. full-scale,
with a Beckman GC-4 gas chromatograph. 0.2 Microliters of the test
mixture was injected at an initial column temperature of
100.degree.C., and the temperature of the column was continuously
increased at 1.33.degree.C. per minute. The versatility of this
column permits the separation of both low boiling compounds
(hexane) and very high boiling compounds (di-normal
butyl-phthalate). The "bleed" of this "ether" bonded-phase column
at high temperatures is minimal, as evidenced by the slight
increase in baseline of the chromatogram approaching 300.degree.C.
for this uncompensated single column system.
The unique selectivity of the chemically bonded polymeric "ether"
packing for gas chromatographic separations is illustrated in FIG.
9. The upper curve shows the separation of a mixture of aliphatic
hydrocarbons and 4-bromobiphenyl on a column of 1.1 percent bonded
"ether" on "Zipax" support operated at 275.degree.C. The lower
curve is the same mixture chromatographed under identical
conditions on a 1.0 percent "Carboxwax" 20M (aliphatic polyether)
column, except that the temperature was 200.degree.C. The
separation factors were very high for these compounds on the
"ether" bonded-phase column operated at 275.degree.C., compared to
those on "Carbowax" 20M at 200.degree.C. A similar pattern is
apparent for the aromatic compound, 4-bromobiphenyl; its retention
time on the "ether" column at 275.degree.C. is 1.4 minutes, as
compared to 0.25 min. for the "Carbowax" 20M column at the same
temperature.
EXAMPLE 5
Preparation of ".beta.Cyanoethyl"/"Zipax"
Bonded-phase for Gas Chromatography
To a 100 ml. round-bottom flask is added 25 ml. of reagent grade
dioxane, 2 ml. of 0.1N hydrochloric acid, and 5.0 ml. of a 70
mg./ml. solution of General Electric XC-3711
(.beta.-cyanoethyltriethoxysilane) in dioxane. This mixture is
refluxed under a condenser for 1 hour and is then added to a
shallow evaporating dish containing 25 g. of 100-120 mesh
acid-treated "Zipax." The solvent is removed from the mixture by
stirring under a stream of warm air from a heat gun. The mixture is
then heated at 150.degree.C. for one hour in a circulating air
oven. The resulting material is then extracted three times by
refluxing in 300 ml. volumes of absolute methanol as described in
earlier examples. Elemental analysis of the final material showed
0.205, 0,205 percent carbon, 0.025, 0.020 percent hydrogen, and
0.076, 0.071 percent nitrogen, indicating 0.72 percent polymer on
the surface of the "Zipax," or 86 percent of theory.
The very high temperature stability of the ".beta.-cyanoethyl"
bonded-phase packing is illustrated in FIG. 10. This figure shows
the background current of a flame ionization detector operated at 1
.times. 10.sup.-.sup.9 amp., full-scale, when a 1/4 inch o.d.
.times. 1/8 inch i.d. glass column of ".beta.-cyanoethyl"
bonded-phase was programmed from 100.degree. to 300.degree.C. On
the same plot is the background current obtained under the same
conditions (slightly displaced upward on the scale to show
differences) for a similar column of 1 percent General Electric
XE-60 (25 percent cyanoethyl, methyl silicon polymer), mechanically
dispersed on 100-120 mesh "Zipax" support. Both of these columns
were conditioned at 250.degree.C. for 16 hours before this test.
While the bonded "nitrile" packing shows essentially no "bleed" at
275.degree.C., the conventional XE-60 column starts to show
significant background at about 225.degree.C. The "nitrile" column
is stable to at least 300.degree.C., while the XE-60 packing cannot
be used to this temperature because of the high level of
"bleed."
The thermal stability of the chemically-bonded "nitrile" stationary
phase is further evidenced by the thermogravimetric plot shown in
FIG. 11. These data were obtained on a Du Pont Model 950
Thermogravimetric Analyzer (E. I. du Pont de Nemours & Co.,
Inc., Wilmington, Del.) operated at a heating rate of 10.degree.C.
per minute, using a flow of 85 cc./min. of air. The curve shows the
weight loss on a 100 mg. sample of the "nitrile" material as a
function of temperature. This study indicates that the organic
polymeric phase is essentially stable to about 325.degree.C., then
begins to degrade slowly at higher temperatures.
The level of column "bleed" at 300.degree.C. for the "ether" and
"nitrile" bonded-phase materials is compared in Table III with
conventional mechanically-held liquid phases. "Zipax" was used as
the support in all measurements, and all columns were conditioned
at 250.degree.C. for 16 hours prior to taking the measurements,
except "Carbowax" 20M, which was conditioned at 200.degree.C. The
selectivity of the "ether" bonded-phase packing can be roughly
compared to the aliphatic polyether, "Carbowax" 20M. The data in
Table III demonstrate the superior thermal stability of both
"Carbowax" 20M and the "ether" bonded-phase on "Zipax" support. A
similar comparison is made between the bonded "nitrile" material
and silicone XE-60, a polymer containing 25 percent nitrile
groupings. The superior thermal stability of the bonded polymer is
apparent. An even lower level of column "bleed" occurs at a lower
polymer concentration, as indicated by the data for the 0.12
percent "nitrile" polymer column.
TABLE III
"BLEED" RATES OF GAS CHROMATOGRAPHIC COLUMNS
Support - 100-140 mesh
"Zipax" Weight Percent "Bleed" Rate at 300.degree.C. Stationary
Phase Loading (Amperes, full-scale)
__________________________________________________________________________
"Carbowax" 20M 1.0 5 .times. 10.sup.-.sup.10 "Ether" Bonded-phase
1.1 7 .times. 10.sup.-.sup.10 XE-60 Silicone 1.0 2 .times.
10.sup.-.sup.9 "Nitrile" Bonded-phase 0.72 1 .times.
10.sup.-.sup.10 "Nitrile" Bonded-phase 0.12 1 .times.
10.sup.-.sup.11
__________________________________________________________________________
The chemically bonded "nitrile" stationary phase exhibits very high
specific retentions for polar compounds, compared to similar
conventional GC stationary phases; the elution temperatures of
polar compounds chromatographed on the "nitrile" bonded-phase
packing by programmed temperature gas chromatography are large.
This characteristic is a function of the high polarity of the
polymeric phase, and may be advantageous for carrying out certain
selective separations. FIG. 12 shows the separation of a mixture of
aromatic hydrocarbons carried out on a column containing only 0.12
percent "nitrile" bonded-phase on "Zipax." After sample injection,
the column was held at 25.degree.C. for 1 minute, then programmed
at 15.degree.C./min. to 100.degree.C.
The stability of the bonded-phase packings at high temperatures
makes it possible for chromatographic separations to be carried out
over wide ranges in temperature without changes occurring to the
column. With the use of the present invention, it is possible to
prepare chromatographic phases of very high polarity which at high
temperatures demonstrate a stability previously obtained only with
nonpolar silicone stationary phases (polydimethylsiloxane,
polymethylphenylsiloxane, etc.).
EXAMPLE 6
Ion Exchange Bonded-Phase
for Liquid Chromatography
Fifty milliliters of a 10 percent (by weight) aqueous solution of
Union Carbide A-1100 silane (.gamma.-aminoprolyltriethoxysilane)
which has been standing for 2 hours at room temperature is placed
in a wide-mouthed jar with 25 grams of acid-treated "Zipax,"
<400 mesh. The solution is then adjusted to about pH 6 with
dilute acetic acid. A vacuum (water pump) is repeatedly pulled on
the solution to degas the support thoroughly and allow the solution
to completely wet the support surface. The mixture is transferred
to a sintered glass Buchner filter funnel and the excess solution
filtered off. The moist bed is transferred to an evaporating dish
and heated at 125.degree.C. for 1 hour in a circulating air oven.
The resulting sample is refluxed three times with fresh 200 ml.
portions of absolute methanol, each time decanting the solvent
after the treatment. The extracted beads are filtered off on a
sintered glass Buchner funnel, air-dried, and then heated in a
circulating air oven at 125.degree.C. for 30 minutes.
This weak anion exchange packing can be used to separate a wide
variety of acidic compounds, or other materials which are retained
on this basic chromatographic medium. The amino functionality can
also be quaternized to a strongly basic tetraalkylammonium
derivation by well-known organic reactions. The quaternized form is
a useful strong anion exchanger.
EXAMPLE 7
Bonded-Phase for Capillary
Gas Chromatography
A 100 meter 0.01 inch i.d. glass capillary is cleaned by passing
concentrated nitric acid through the tubing while heating over a
steam bath. The capillary is then thoroughly washed with distilled
water to eliminate all acid, rinsed with reagent grade acetone and
dried with dry nitrogen. About 50 ml. of a 5 percent (by weight)
solution of Union Carbide Silane A-16 (amyltriethoxysilane) is
passed through the capillary, dry nitrogen connected to the tubing,
and the excess solution removed by the pressure of the gas. The
flow of dry nitrogen is continued through the capillary until all
of the excess solvent has been evaporated, leaving a thin film of
A-16 on the interior surface of the glass. A stream of moist air
(relative humidity of about 85 percent) is then passed through the
capillary until the silane ester is completely hydrolyzed, as
evidenced by no more ethanol being evolved from the tubing. The
capillary is then placed in a 110.degree.C. oven and a stream of
dry nitrogen slowly passed through the tubing for several
hours.
This capillary bonded-phase column is particularly useful for
separating complex mixtures of aliphatic and substituted aromatic
hydrocarbons.
EXAMPLES 8-12
Given in Table IV are a number of bonded-phase chromatographic
packings prepared by the techniques and from the reagents shown.
Also given are the applications for which these systems may be
used. ##SPC6##
* * * * *