Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
IMPROVED LIQUID CHROMATOGRAPHIC MEDIA FOR
POLYNUCLEOTIDE SEPARATION
FIELD OF THE INVENTION
The present invention is directed to the separation of polynucleotides
using nonporous beads. More specifically, the invention is directed to the
chromatographic separation of both single stranded and double stranded
polynucleotides by chromatography using chromatographic columns
containing nonporous beads, where the beads comprise either organic or
inorganic particles which are coated with a polymer, or non-polar substituted
polymer, and/or which have substantially all surface substrate groups
substituted with a non-polar hydrocarbon or non-ionic substituted
hydrocarbon.
BACKGROUND OF THE INVENTION
Separations of pofynucleotides such as DNA have been traditionally
performed using slab gel electrophoresis or capillary electrophoresis.
However, liquid chromatographic separations of pofynucleotides are
becoming more important because of the ability to automate the analysis and
to collect fractions after they have been separated. Therefore, columns for
polynucleotide separation by liquid chromatography (LC) are becoming more
important.
Silica-based columns are by far the most common LC columns. Of
these, reverse phase silica-based columns are preferred because they have
high separation efficiencies, are mechanically stable, and a variety of
functional groups can be easily attached for a variety of column
selectivities.
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Although silica-based reverse phase column materials have performed
adequately for separating single stranded DNA, these materials have not
performed well for separating double stranded DNA. The peaks from double
stranded DNA separations using silica-based materials are badly shaped or
broad, or the double stranded DNA may not even elute. Separations can
take up to several hours, or the resolution, peak symmetry, and sensitivity of
the separation are poor.
High quality materials for DNA separations have been based on
polymeric substrates, as disclosed in U.S. Patent No. 5,585,236. There
exists a need for silica-based column packing material and other materials
that are suitable for separation of double stranded DNA.
SUMMARY OF THE INVENTION
Accordingly, one object of the present invention is to provide a
chromatographic method for separating polynucleotides with improved
separation and efficiency.
This and other objects of the invention, which will become apparent
from reading the following specification, have been achieved by the method of
the present invention in which polynucfeotides are chromatographically
separated using a column containing nonporous beads, where the beads
comprise either organic or inorganic particles which are coated with a
polymer, or non-polar substituted polymer, and/or which have substantially all
surface substrate groups substituted with a non-polar hydrocarbon or non-
ionic substituted hydrocarbon.
Disclosed herein is a method for separating a mixture of
poiynucieotides. The method comprises flowing a mixture of poiynucleotides
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_. ........._ ..._..__....._.. .. _ _~ _ _.. _ .. ..___._..»~ __......._
___...
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having up to 1500 base pairs through a separation column containing beads
having an average diameter of 0.5 to 100 microns, and separating the mixture
of polynucleotides. The beads comprise nonporous particles coated with a
hydrocarbon or non-polar substituted polymer or having substantially all
surface substrate groups reacted with a non-polar hydrocarbon or substituted
hydrocarbon group. Precautions are taken during the production of the beads
so that they are substantially free of multivalent cation contaminants and the
beads are treated, for example by an acid wash treatment, to remove any
residual surface metal contaminants. In one embodiment, the beads are
characterized by having a DNA Separation Factor (as defined hereinbelow) of
at least 0.05. In a preferred embodiment, the beads are characterized by
having a DNA Separation Factor of at least 0.5.
The separation is preferably by Matched Ion Pofynucleotide
Chromatography (MIPC) as defined hereinbelow. The beads preferably have
an average diameter of about 1 - 5 microns. The nonporous particle is
preferably selected from silica, silica carbide, silica nitrite, titanium
oxide,
aluminum oxide, zirconium oxide, carbon, insoluble polysaccharides such as
cellulose, and diatomaceous earth, or any of these materials that have been
modified to be nonporous. The nonporous particle is most preferably silica,
which preferably is substantially free from unreacted silanol groups. The
particles can be prepared by non-covalently bonded coatings, covalently
bonded coatings, or reaction of the silanol groups with hydrocarbon groups.
The nonporous particle can be coated with a polymer. The polymer is
preferably selected from polystyrenes, polymethacrylates, polyethylenes,
polyurethanes, polypropylenes, polyamides, cellulose, polydimethyl siloxane,
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and polydialkyl siloxane. The polymer is optionally unsubstituted or
substituted with hydrocarbon groups or other groups having nonionic
substituents. The polymer can be optionally substituted with hydrocarbon
groups having from 1 to 1,000,000 carbons, the hydrocarbon groups
optionally being alkyl groups with from 1 to 100 carbons and preferably from 1
to 24 carbons. Hydrocarbon groups from 24 to 1,000,000 are described
herein as hydrocarbon polymers and have the constituency of hydrocarbon
groups as defined herein.
The reaction of organosilanols (e.g. HO-Si-R3) or alkoxy- (e.g., RO-Si-
R3) silanes with silica supports without polymerization can also produce good
packings. The method produces a dense monolayer of functional groups of
alkyl or alkylsubstituted, ester, cyano, and other nonionic groups. The use of
monofunctional dimethyl silanes (X-Si(CH3)2-R) provides a homogeneous
organic coating with a minimum of residual Si-OH groups. Monochlorosilane
reagents are preferred, if the required organic functionality can be prepared.
These reactions are reproducible and provide high quality packing materials.
Unreacted, accessible silanols can be left after the initial reaction. The
nonporous particle is preferably endcapped with a tri(lower alkyl)chlorosilane
(preferably a trimethylchlorosilane) to block residual reactive sifanol sites
following the coating or hydrocarbon substitution. Alternatively, all of the
silanol sites can be reacted with an excess of the endcapping reagent to
extinguish all reactive silanol groups. Endcapping of the nonporous particle
can be effected by reaction of the nonporous particle with the corresponding
hydrocarbon substituted silane, such as trialkyl chiorosilane (eg. trimethyl
chlorosilane) or by reaction with the corresponding hydrocarbon substituted
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disilazane, such as dichloro-tetraalkyl-disilazane (eg. dichloro-tetramethyl-
disilazane).
The method of the present invention can be used to separate double
stranded polynucleotides having up to about 1500 to 2000 base pairs. In
many cases, the method is used to separate polynucleotides having up to
600 bases or base pairs, or which have up to 5 to 80 bases or base pairs.
The method is performed at a temperature within the range of 20°C
to
90°C to yield a back-pressure not greater than 10,000 psi. The method
also
preferably employs an organic solvent that is water soluble. The solvent is
preferably selected from the group consisting of alcohols, nitrites,
dimethylformamide, esters, and ethers. The method also preferably employs
a counter ion agent selected from trialkylamine acetate, trialkylamine
carbonate, and trialkylamine phosphate. The most preferred counter ion
agent is triethylammonium acetate or triethylammonium hexafluoroisopropyl
alcohol.
The method also preferably comprises supplying and feeding solutions
entering the separation column with components having process solution-
contacting surfaces which contact process solutions held therein or flowing
therethrough. The process solution-contacting surfaces are material which
does not release multivalent cations into aqueous solutions held therein or
flowing therethrough, so that the column and its contents are protected from
multivalent cation contamination The process solution-contacting surfaces
are preferably material selected from the group consisting of titanium, coated
stainless steel, passivated stainless steel, and organic polymer. Multivalent
cations in eluent solutions and sample solutions entering the column are also
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preferably removed by contacting the solutions with multivalent cation capture
resin before the solutions enter the column so as to protect the resin bed
from
multivalent cation contamination. The multivalent capture resin is selected
from cation exchange resin and chelating resin. The column and process
solutions held therein or flowing therethrough are preferably substantially
free
of multivalent cation contaminants. The polynucleotides are separated by
Matched ion Polynucleotide Chromatography.
Also disclosed herein is a method for separating a mixture of
polynucleotides, comprising flowing a mixture of polynucfeotides having up to
1500 base pairs through a separation column containing beads having an
average diameter of 0.5 to 100 microns, and separating the mixture of
polynucleotides by Matched Ion Polynucleotide Chromatography. The beads
comprise nonporous particles coated with a polymer or having substantially
all surface substrate groups reacted and/or endcapped with a non-polar
hydrocarbon or substituted hydrocarbon group. The beads are characterized
by having a DNA Separation Factor of at least 0.05. The column and process
solutions held therein or flowing therethrough are substantially free of
multivalent cation contaminants. The method is performed at a temperature
within the range of 20°C to 90°C to yield a back-pressure not
greater than
10,000 psi. An organic solvent that is water soluble is used in the
performance of the method.
Also disclosed herein is a bead comprising a nonporous particle
coated with a polymer. The bead has an average diameter of 0.5 to 100
microns and is characterized by having a DNA Separation Factor of at least
0.05. In a preferred embodiment, the bead is characterized by having a DNA
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Separation Factor of at least 0.5. The bead preferably has a diameter of
about 1 - 5 microns. The nonporous particle is preferably selected from
silica,
silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium
oxide,
carbon, insoluble polysaccharides such as cellulose, and diatomaceous earth,
or any of these materials that have been modified to be nonporous. The
nonporous particle is most preferably silica, which preferably has minimum
silanol groups. The polymer is preferably selected from polystyrene,
polymethacrylate, polyethylene, polyurethane, polypropylene, polyamide,
cellulose, polydimethyl siioxane, and polydialkyl siloxane, and is preferably
unsubstituted, alkylated, or alkyl or aryl substituted, or alkylated with a
substituted alkyl group methyl-substituted, or ethyl-substituted. The polymer
can be alkylated with alkyl groups having 1 - 22 carbon atoms, preferably, 8 -
18 carbon atoms.
Also disclosed herein is a bead comprising a nonporous particle having
substantially all surface substrate groups reacted with a hydrocarbon group
and then endcapped with a non-polar hydrocarbon or substituted
hydrocarbon group, preferably a tri(lower alkyi)chlorosilane or tetra(lower
alkyl)dichlorodisilazane. The bead has an average diameter of 0.5 to 100
microns and is characterized by having a DNA Separation Factor of at least
0.05. The bead preferably has a diameter of about 1 - 5 microns.
The nonporous particle is preferably selected from silica, silica carbide,
silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon,
insoluble polysaccharides such as cellulose, and diatomaceous earth, or any
of these materials that have been modified to be nonporous. The nonporous
particle is most preferably silica, which preferably has minimum silanol
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groups. Endcapping of the nonporous particle can be effected by reaction of
the nonporous particle with trimethyl chlorosilane or dichloro-tetraisopropyl-
disilazane.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of how the DNA Separation
Factor is applied to a separation.
FIG. 2 is a schematic drawing of a cross-section of a representation of
a reverse phase bead with a silica core and endcapping shielding.
FIG. 3 is a schematic drawing of a cross-section of a representation of
a reverse phase bead with a silica core and polymer shielding.
FIG. 4 is a M1PC separation of pUCl8 DNA-Haelll digestion fragments
on a column containing alkylated polystyrene-divinylbenzene) beads. Peaks
are labeled with the number of base pairs of the eluted fragment.
FIG. 5 is a MIPC separation of pUCl8 DNA-Haelll digestion fragments
on a column containing nonporous 2.1 micron beads of underivatized
poiy(styrene-divinylbenzene).
FIG. 6 is a Van't Hoff plot of the retention factor 1/T ~°K -'J with
alkylated
polystyrene-divinylbenzene) beads showing positive enthalpy using
acetonitrile as the solvent.
FIG. 7 is a Van't Hoff plot of the retention factor 1/T (°K ~'J
with
underivatized polystyrene-divinylbenzene) beads showing positive enthalpy
using acetonitrile as the solvent.
FIG. 8 is a Van't Hoff plot of the retention factor 1/T ~°K-'J with
alkylated
polystyrene-divinylbenzene) beads showing negative enthalpy using
methanol as the solvent.
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FIG. 9 is a separation using alkyiated beads and acetonitrile as
solvent.
FIG. 10 is a separation using alkylated beads and 50.0% methanol as
the solvent.
FIG. 11 is a separation using alkylated beads and 25.0% ethanol as
the solvent.
FIG. 12 is a separation using alkylated beads and 25.0% vodka
(Stofichnaya, 100 proof) as the solvent.
FIG. 13 is a separation using alkylated beads and 25.0% 1-propanol as
the solvent.
FIG. 14 is a separation using alkylated beads and 25.0% 1-propanol as
the solvent.
FIG. 15 is a separation using alkylated beads and 10.0% 2-propanol as
the solvent.
FiG. 1 fi is a separation using alkylated beads and 10.0% 2-propanol as
the solvent.
FIG. 17 is a separation using alkylated beads and 25.0% THF as the
solvent.
FIG. 18 is an isocratic/gradient separation on non-alkylated
polystyrene-divinylbenzene) beads.
DETAILED DESCRIPTION OF THE INVENTION
In its most general form, the subject matter of the present invention is
the separation of poiynucleotides by Matched Ion Polynucleotide
Chromatography utilizing columns filled with nonporous beads having an
average diameter of about 0.5 -100 microns; preferably, 1 - 10 microns; more
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preferably, 1 - 5 microns. Beads having an average diameter of 1.0 - 3.0
microns are most preferred.
In U.S. Patent No. 5,585,236, Bonn et al. had characterized the
polynucleotide separation process as reverse phase ion pairing
chromatography (RPIPC). However, since RPIPC does not incorporate
certain essential characteristics described in the present invention, another
term, Matched Ion Polynucleotide Chromatography (MIPC), has been
selected. MIPC as used herein, is defined as a process for separating single
and double stranded polynucleotides using non-polar beads, wherein the
process uses a counter ion agent, and an organic solvent to desorb the
polynucleotide from the beads, and wherein the beads are characterized as
having a DNA Separation Factor of at least 0.05. In a preferred embodiment,
the beads are characterized as having a DNA Separation Factor of at least
0.5.
The performance of the beads of the present invention is demonstrated
by high efficiency separation by MIPC of double stranded and single stranded
DNA. We have found that the best criterion for measuring performance of the
beads is a DNA Separation Factor. This is measured as the resolution of
257- and 267-base pair double stranded DNA fragments of a pUC18 DNA-
Haelll restriction digest wherein the distance from the valley between the
peaks to the top of one of the peaks, over the distance from the baseline to
the valley. Referring to the schematic representation of FlG. 1, the DNA
Separation Factor is determined by measuring the distance "a" from the
baseline to the valley "e" between the peaks "b" and "c" and the distance "d"
from the valley "e" to the top of one of the peaks "b" or "c". If the peak
heights
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are unequal, the highest peak is used to obtain "d." The DNA Separation
Factor is the ratio of d/(a+d). The peaks of 257- and 267-base pairs in this
schematic representation are similar in height. Operational beads of the
present invention have a DNA Separation Factor of at least 0.05. Preferred
beads have a DNA Separation Factor of at least 0.5. In an optimal
embodiment, the beads have a DNA Separation Factor of at least 0.95.
Without wishing to be bound by theory, we believe that the beads
which conform to the DNA Separation Factor as specified herein have a pore
size which essentially excludes the polynucleotides being separated from
entering the bead. As used herein, the term "nonporous" is defined to denote
a bead which has surface pores having a diameter that is less than the size
and shape of the smallest DNA fragment in the separation in the solvent
medium used therein. Included in this definition are beads having these
specified maximum size restrictions in their natural state or which have been
treated to reduce their pore size to meet the maximum effective pore size
required. Preferably, all beads which provide a DNA Separation Factor of at
least 0.05 are intended to be included within the definition of "nonporous"
beads.
The surface conformations of nonporous beads of the present
invention can include depressions and shallow pit-like structures which do not
interfere with the separation process. A pretreatment of a porous bead to
render it nonporous can be effected with any material which will fill the
pores
in the bead structure and which does not significantly interfere with the MIPC
process.
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Pores are open structures through which eluent and other materials
can enter the bead structure. Pores are often interconnected so that fluid
entering one pore can exit from another pore. We believe that pores having
dimensions that allow movement of the polynucieotide into the interconnected
pore structure and into the bead impair the resolution of separations or
result
separations that have very long retention times. In MIPC, however, the beads
are "nonporous" and the polynucleotides do not enter the bead structure.
The term polynucleotide is defined as a linear polymer containing an
indefinite number of nucleotides, linked from one ribose (or deoxyribose) to
another via phosphoric residues. The present invention can be used in the
separation of RNA or of double- or single-stranded DNA. For purposes of
simplifying the description of the invention, and not by way of limitation,
the
separation of double-stranded DNA will be described in the examples herein,
it being understood that all polynucleotides are intended to be included
within
the scope of this invention.
Chromatographic efficiency of the column beads is predominantly
influenced by the properties of surface and near-surface areas. For this
reason, the following descriptions are related specifically to the close-to-
the-
surface region of the polymeric beads. The main body and/or the center of
such beads can exhibit entirety different chemistries and sets of physical
properties from those observed at or near the surface of the polymeric beads
of the present invention.
The beads of the invention comprise a nonporous particle which has
non-polar molecules or a non-polar polymer attached to or coated on its
surface. In general, the beads comprise nonporous particles which have
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been coated with a polymer or which have substantially all surface substrate
groups reacted with a non-polar hydrocarbon or substituted hydrocarbon
group, and any remaining surface substrate groups endcapped with a
tri(lower alkyl)chlorosilane or tetra(lower alkyl)dichlorodisilazane as
described
above.
The nonporous particle is preferably an inorganic particle, but can be a
nonporous organic particle. The nonporous particle can be, for example,
silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide,
zirconium
oxide, carbon, insoluble polysaccharides such as cellulose, or diatomaceous
earth, or any of these materials which have been modified to be nonporous.
Examples of carbon particles include diamond and graphite which have been
treated to remove any interfering contaminants. The preferred particles are
essentially non-deformable and can withstand high pressures. The nonporous
particle is prepared by known procedures. The preferred particle size is
about 0.5 -100 microns; preferably, 1 - 10 microns; more preferably, 1 - 5
microns. Beads having an average diameter of 1.0 - 3.0 microns are most
preferred.
Because the chemistry of preparing conventional silica-based reverse
phase HPLC materials is well-known, most of the description of the beads of
the invention herein is presented in reference to silica. It is to be
understood,
however, that other nonporous particles, such as those listed above, can be
modified in the same manner and substituted for silica in the process of the
invention. For a description of the general chemistry of silica, see Poole,
Colin F. and Salwa K. Poole, Chromatography Today, Elsevier:New York
(1991 ), pp. 313-342 and Snyder, R. L. and J. J. Kirkland, Introduction to
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Modern Liquid Chromatography, 2"° ed., John Wiiey & Sons, Inc.:New
York
(1979), pp. 272-278, the disclosures of which are hereby incorporated herein
by reference in their entireties.
The nonporous beads of the invention are characterized by having
minimum exposed silanol groups after reaction with the coating or alkylation
reagents. Minimum silanol groups are needed to reduce the interaction of the
DNA with the substrate and also to improve the stability of the material in a
high pH and aqueous environment. Silanol groups can be harmful because
they can repel the negative charge of the DNA molecule, preventing or
limiting the interaction of the DNA with the stationary phase of the column.
Another possible mechanism of interaction is that the silanof can act as ion
exchange sites, taking up metals such as iron (III) or chromium (111). Iron
(III)
or other metals which are trapped on the column can distort the DNA peaks
or even prevent DNA from being eluted from the column.
Silanol groups can be hydrolyzed by the aqueous-based eluent.
Hydrolysis will increase the polarity and reactivity of the stationary phase
by
exposing more silanol sites, or by exposing metals that can be present in the
silica core. Hydrolysis will be more prevalent with increased silanol groups.
The effect of silanol groups on the DNA separation depends on which
mechanism of interference is most prevalent. For example, iron (III) can
become attached to the exposed silanol sites, depending on whether the iron
(III) is present in the eluent, instrument or sample.
The effect of metals can only occur if metals are already present within
the system or reagents. Metals present within the system or reagents can get
trapped by ion exchange sites on the silica. However, if no metals are
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present within the system or reagents, then the silanol groups themselves can
cause interference with DNA separations. Hydrolysis of the exposed sifanol
sites by the aqueous environment can expose metals that might be present in
the silica core.
Fully hydrolyzed silica contains a concentration of about 8 Nmoles of
silanoi groups per square meter of surface. At best, because of steric
considerations, a maximum of about 4.5 Nmoles of silanoi groups per square
meter can be reacted, the remainder of the silanol being sterically shielded
by
the reacted groups. Minimum silanol groups is defined as reaching the
theoretical limit of or having sufficient shield to prevent silanoi groups
from
interfering with the separation.
Numerous methods exist for forming nonporous silica core particles.
For example, sodium silicate solution poured into methanol will produce a
suspension of finely divided spherical particles of sodium silicate. These
particles are neutralized by reaction with acid. In this way, globular
particles
of silica gel are obtained having a diameter of about 1 - 2 microns. Silica
can
be precipitated from organic liquids or from a vapor. At high temperature
(about 2000°C), silica is vaporized, and the vapors can be condensed to
form
finely divided silica either by a reduction in temperature or by using an
oxidizing gas. The synthesis and properties of silica are described by R. K.
Iler in The Chemistry of Silica, Solubility, Polymerization, Colloid and
Surface
Properties, and Biochemistry, John Wiley & Sons:New York (1979).
W. Stober et al. described controlled growth of monodisperse silica
spheres in the micron size range in J. Colloid and Interface Sci., 26:62-69
(1968). Stober et al. describe a system of chemical reactions which permit
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the controlled growth of spherical silica particles of uniform size by means
of
hydrolysis of alkyl silicates and subsequent condensation of silicic acid in
alcoholic solutions. Ammonia is used as a morphological catalyst. Particle
sizes obtained in suspension range from less than 0.05 Nm to 2 Nm in
diameter.
Nonporous silica core beads can be obtained from Micra Scientific
(Northbrook, IL) and from Chemie Uetikkon (Lausanne, Switzerland).
To prepare the nonporous beads of the invention, the nonporous
particle is coated with a polymer or reacted and endcapped so that
substantially all surface substrate groups of the nonporous particle are
blocked with a non-polar hydrocarbon or substituted hydrocarbon group. This
can be accomplished by several methods.
The organic bonded-phase siloxane coating can be made as a
monomolecuiar layer or as a polymerized multilayer coating. Packings with
so-called monomolecular organic layers are normally prepared by reacting the
surface silanoi groups of siliceous-base particles with mono-, di-, or
trifunctional chloro-, dimethyl-, amino-, siloxy-, or alkoxy-silanes. Typical
monofunctional reactants used in these reactions include X-Si-R , where X =
CI, OH, OCH3, or OCZHS, and R is an organic radical. FIG. 2 is a schematic
representation of a bead 20 having a silica core 22 and a monomolecular
organic layer. (The figure does not necessarily reflect the morphology or pore
structure of the beads of the invention and is meant for illustrative purposes
only.)
Using bi- and trifunctional reactants, such as R2SiX2 and RSiX3, for the
surface modifications, up to two Si-X groups per bonded functional group
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remain unreacted. After treatment with water, hydrolysis of these unreacted
groups takes place, and additional silanol groups are formed (sometimes in a
polymer matrix) in about the same concentration as the bonded organic
functional groups present in the packing. These acidic organo-silanol groups
can significantly affect the retention behavior of solutes and adversely
influence the stability of the packing in aqueous solutions at pH > 7.
Thus, incomplete reaction of the surface with the silane reagent, or the
formation of new Si-OH groups from using bi- or trifunctional modifiers, can
result in a population of residual acidic Si-OH groups that are readily
accessible to molecules of the mobile phase or sample. Therefore, the recent
trend is toward (a) a dense monolayer of functional groups instead of partial
coverage and (b) the use of monofunctional dimethylsilanes [X Si(CH3)2 RJ to
provide a homogeneous organic coating with a minimum possibility of
residual Si-OH groups. Monochlorosilane reagents are preferred, if the
required organic functionality can be prepared. If two of the R groups in the
monofunctional modifier are methyl, surface coverage can be as high as
about 4 pmoles per square meter of organic (based on carbon analysis). in
the latter case, residual Si-OH groups on the silica surface are unavailable
for
chromatographic interactions with most solutes because of steric shielding.
The reaction of organosilanols (e.g., HO-Si-R3) or organoalkoxy- (e.g.,
RO-Si-R3) silanes with silica supports without polymerization can also produce
good packings. These reactions are relatively reproducible, provided that
traces of water or other reactive species are absent. Unreacted, accessible
silanols can be left after the initial reaction, but these can be removed by
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capping of the packing with chlorotrimethylsilane (providing the R groups do
not react with the latter silane).
According to one method, the nonporous particle is coated with a
polymer coating. Suitable polymers for use in coating the particle include
chain reaction polymers and step reaction polymers, for example,
polystyrene, polymethacrylate, polyethylene, polyurethane, polypropylene,
polyamide, insoluble polysaccharides such as cellulose, polydimethyl
siloxane, polydialkyl siloxane, and related materials. The polymer coating can
be attached to the nonporous particle by means of a multi-coating process so
that complete shielding of the surface is achieved.
In the last few years, new bonded phase packings, known as polymer-
coated or polymer-encapsulated packings, have been introduced based on
techniques used to prepare immobilized stationary phases for open tubular
column gas chromatography. In this case, the phases are prepared by
mechanically coating either bare silica or presilanized silica microparticles
with a poly(siioxane) or poly(butadiene} prepolymer, which is then
immobilized by peroxide, azo-tert-butane, or gamma radiation-induced
chemical crosslinking reactions. FIG. 3 is a schematic illustration of a
coated
bead 30 having a silica core 32 and polymer coating 34. (The figure does not
necessarily reflect the morphology or pore structure of the beads of the
invention and is meant for illustrative purposes only.}
An alternative method comprises a combination of covalent bonding
with a vinyl-containing silane molecule and then polymerizing a coating on the
surface of the particles. A second coating can be applied if residual silanol
~~groups or metal groups are present.
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In a variation of this method, the silica surface is first modified by
reaction with vinyltrichlorosilane,nfollowed by polymerizing acrylic acid
derivatives to and over the derivatized silica surface. The availability of a
large number of useful monomers and prepoiymers has enabled a wide
variety of reverse phase, polar, and ion exchange packings to be prepared
using the same general reaction. Also, since the general approach does not
depend on the chemistry of the underlying substrate, materials other than
silica, for example, alumina and zirconia, can be modified and used under
conditions for which silica is unsuitable, for example, with mobile phases
outside the pH range 2 - 7.5. Returning to silica, presiianization decreases
the number of active siianol groups, which are then further shielded by the
polymeric film anchored over the surtace. In reverse phase liquid
chromatography, these packings have shown improved chromatographic
properties compared to monomeric, chemically bonded phases for the
separation of basic solutes. Polymer-encapsulated packings have a film
thickness of about 1 nm to maintain reasonable mass transfer characteristics.
A description of the this procedure has been published by H. Engelhart et al.
(Chromatographia, 27:535 (1989)).
The polymer-coated beads prepared according to either of the above
methods can be used in their unmodified state or can be modified by
substitution with a hydrocarbon group. Any hydrocarbon group is suitable.
The term "hydrocarbon" as used herein is defined to include alkyl and alkyl
substituted aryl groups, having from 1 to 1,000,000 carbons, the alkyl groups
including straight chained, branch chained, cyclic, saturated, unsaturated
nonionic functional groups of various types including, aldehyde, ketone,
ester,
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ether, alkyl groups, and the like, and the aryl groups including as
monocyclic,
bicyclic, and tricyclic aromatic hydrocarbon groups including phenyl,
naphthyl,
and the like. Methods for hydrocarbon substitution are conventional and well-
known in the art and are not an aspect of this invention. The hydrocarbon can
also contain hydroxy, cyano, vitro groups, or the like which are considered to
be non-polar, reverse phase functional groups. The preferred hydrocarbon
groups are alkyl groups, and the description of suitable substitution
processes
hereinbeiow are presented as alkylation for purposes of simplification and not
by way of limitation, it being understood that aryl substitution by
conventional
procedures are also intended to be included within the scope of this
invention.
The polymer-coated beads can be alkyfated by reaction with the
corresponding alkyl halide such as the alkyl iodide. Alkylation is achieved by
mixing the polymer-coated beads with an alkyl halide in the presence of a
Friedel-Crafts catalyst to effect electrophilic aromatic substitution on the
aromatic rings at the surface of the polymer blend. Suitable Friedel-Crafts
catalysts are well-known in the art and include Lewis acids such as aluminum
chloride, boron trifluoride, tin tetrachloride, etc. Substitution with
hydrocarbon
groups having from 1 to 1,000,000 and preferably from 1 to 22 carbons can
be effected by these processes. Hydrocarbon groups having from 23 to
1,000,000 carbons are referenced herein as hydrocarbon polymers.
Alkylation can be accomplished by a number of known synthesis
procedures. These include Friedel-Crafts alkylation with an alkyl halide,
attachment of an alkyl alcohol to a chloromethylated bead to form an ether,
etc. Although the preferred method for alkylating the polymer-coated beads
of the present invention is alkylation after the polymer coating has been
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formed on the nonporous particle, an alternative method of alkylation is to
polymerize alkylated monomers to form an alkylated polymer coating on the
nonporous particle. In this embodiment, the monomers will be substituted
with alkyl groups having any number of carbon atoms, for example, from 1 to
100, 1 to 50 or 1 to 24, for example, depending upon the requirements of the
separation variables.
As an alternative to polymer coating, the nonporous particle can be
functionalized with an alkyl group or other non-polar functional group
including cyano, ester, and other non-ionic groups, followed by a complete
IO endcapping process to reduce silanol and metal interaction. Endcapping of
the nonporous particle can be achieved by reacting the particle with trialkyl
chlorosilane or tetraalkyl dichlorodisilazane, such as, for example, trimethyl
chlorosilane or dichloro-tetraisopropyl-disilazane.
A large number of factors influence the success of the bonding
IS reactions and the quality of the final bonded-phase product. The rate and
extent of the bonding reaction depends on the reactivity of the silane, choice
of solvent and catalyst, time, temperature, and the ratio of reagents to
substrate. Reactive organosilanes with C1, OH, OR, N(CH3)2, OCOCF3, and
enolates as leaving groups have been widely used. The dimethyiamine,
20 trifluoroacetate, and enol ethers of pentane-2,4-dione are the most
reactive
leaving groups, although economy, availability, and familiarity result in the
chlorosilanes and alkoxysilanes being the most widely used, particularly
among commercial manufacturers. Initially, reactions can be almost
stoichiometric but, as the surface coverage approaches a maximum value,
25 the reaction becomes very slow. For this reason, reaction times tend to be
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long (12 - 72 hours), reaction temperatures moderately high (in most cases,
around 100°C) and, in the case of chlorosilanes, an acid acceptor
catalyst
(e.g., pyridine) is used. Some reagents, such as the alkylsilyl enolates and
alkylsilyldimethylamines, do not require additional catalyst, or even solvent,
to
carry out the reaction. The most common solvents employed are toluene and
xylene, although other solvents, such as carbon tetrachloride,
trichloroethane,
and dimethylformamide (DMF), have been recommended as being superior.
Since the bonding reactions are carried out by reffuxing in an inert
atmosphere, solvents are often selected based on their capacity to be a good
solvent for the organosilanes and to attain the desired reaction temperature
at
reflex. Except for 3-cyanopropylsiloxane bonded phases, the high reactivity
of chlorosilanes towards certain polar functional groups (e.g., OH, etc.)
precludes the use of these groups for the preparation of polar, reverse phase
bonded phases. Alkoxysilanes containing acidic or basic functional groups
are autocatalytic and the bonded phases are usually prepared by refluxing
the sifane in an inert solvent at a temperature high enough to distill off the
alcohol formed by the condensation reaction with the surface silanoi groups.
Bonding of neutral, polar ligands generally requires the addition of a
catalyst,
such as toluene-4-sulfonic acid or triethylamine, in the presence of
sufficient
water to generate monolayer coverage of the silica. The presence of water
speeds up the hydrolysis of the alkoxy groups of the adsorbed organosilane,
which tends to react with surface silanol groups rather than polymerize in
solution. It seems to be a general problem in the preparation of polar bonded
phases that surface silanoi groups are blocked by physically adsorbed
organosilanes, giving rise to a lower bonded phase density after workup than
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the maximum theoretically predicted. The bonded phase density can be
increased by repeating the reaction a second time or exposed silanol groups
minimized by endcapping.
Although most bonded phases are prepared from organosilanes
containing a single functionalized ligand bonded to silicon, with the
remaining
groups being leaving groups and/or methyl groups, more highly substituted
organosilanes can also be used. Bifunctional organosilanes, such as 1,3-
dichlorotetraisopropyldisilazane, are able to react with surface silanol
groups
at both ends of the chain, forming a bonded phase that is more hydrolytically
stable than bonded phases formed from conventional organosilanes. The
bidentate organosilanes have reactive sites that more closely match the
spacing of the silanol groups on the silica surface and provide a higher
bonded phase coverage than is achieved with dichlorosilanes with both
leaving groups attached to the same silicon atom. For alkyfdimethylsilanes,
increasing the length of the alkyl group increases the hydrolytic stability of
the
bonded phase relative to that of the trimethylsilyl bonded ligands. Increasing
the chain length of the methyl groups increases the hydrolytic stability of
the
bonded phase, but reduces the phase coverage due to steric effects. The
use of monofunctional organosilanes containing one or two bulky groups, for
example, isopropyl or t-butyl, on the silicon atom of the silane can become
more important in the preparation of bonded phases for use at low pH. The
bulky alkyl groups provide better steric protection to the hydrolytically
sensitive siloxane groups on the packing surface than does the methyl group.
The general process of coating and endcapping of a silica substrate is
well-known technology. However, the general understanding of those who
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have used these materials is they are not suitable for high performance
double stranded DNA separations. However, the beads of this invention are
formed by a more careful application of the coating and end-capping
procedures to effect a thorough shielding of the silica core, the resulting
beads having the ability to perform rapid separations of both single stranded
and double stranded DNA which are equal to or better than those achieved
using the afkylated nonporous polymer beads disclosed in U.S. Patent No.
5,585,236, for example.
The beads of the invention are also characterized by having low
amounts of metal contaminants or other contaminants that can bind DNA.
The preferred beads of the present invention are characterized by having
been subjected to precautions during production, including a decontamination
treatment, such as an acid wash treatment, designed to substantially
eliminate any multivalent cation contaminants (e.g. Fe(III), Cr(III), or
colloidal
metal contaminants). Only very pure, non-metal containing materials should
be used in the production of the beads in order that the resulting beads will
have minimum metal content.
Care must be taken during the preparation of the beads to ensure that
the surface of the beads has minimum silanol or metal oxide exposure and
that the surface remains nonporous.
To achieve high resolution chromatographic separations of
polynucleotides, it is generally necessary to tightly pack the chromatographic
column with the solid phase nonporous beads. Any known method of packing
the column with a column packing material can be used in the present
invention to obtain adequate high resolution separations. Typically, a slurry
of
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the beads is prepared using a solvent having a density equal to or less than
the density of the beads. The column is then filled with the bead slurry and
vibrated or agitated to improve the packing density of the beads in the
column. Mechanical vibration or sonification are typically used to improve
packing density.
For example, to pack a 50 x 4.6 mm i.d. column, 2.0 grams of beads
can be suspended in 10 mL of methanol with the aid of sonification. The
suspension is then packed into the column using 50 mL of methanol at 8,000
psi of pressure. This improves the density of the packed bed.
The separation method of the invention is generally applicable to the
chromatographic separation of single stranded and double stranded
polynucleotides of DNA and RNA. Samples containing mixtures of
polynucleotides can result from total synthesis of polynucieotides, cleavage
of
DNA or RNA with restriction endonucleases or with other enzymes or
chemicals, as well as polynucleotide samples which have been multiplied and
amplified using poiymerase chain reaction techniques.
The method of the present invention can be used to separate double
stranded polynucleotides having up to about 1500 to 2000 base pairs. In
many cases, the method is used to separate polynucleotides having up to
600 bases or base pairs, or which have up to 5 to 80 bases or base pairs.
In a preferred embodiment, the separation is by MIPC. The nonporous
beads of the invention are used as a reverse phase material that will function
with counter ion agents and a solvent gradient to produce the DNA
separations. In MIPC, the DNA fragments are matched with a counter ion
agent and then subjected to reverse phase chromatography using the
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nonporous beads of the present invention. Counter ion agents that are
volatile, such as trialkylamine acetate, trialkylamine carbonate,
trialkylamine
phosphate, etc., are preferred for use in the method of the invention, with
triethylammonium acetate (TEAA) and triethylammonium hexafluoroisopropyl
alcohol being most preferred.
To achieve optimum peak resolution during the separation of DNA by
MIPC using the beads of the invention, the method is performed at a
temperature within the range of 20°C to 90°C to yield a back-
pressure not
greater than 10,000 psi. In general, separation of single-stranded fragments
should be performed at higher temperatures.
We have found that the temperature at which the separation is
performed affects the choice of solvents used in the separation. When the
separation is performed at a temperature within the above range, an organic
solvent that is water soluble is preferably used, for example, alcohols,
nitrites,
dimethylformamide (DMF), esters, and ethers. Water soluble solvents are
defined as those which exist as a single phase with aqueous systems under
all conditions of operation of the present invention. Solvents which are
particularly preferred for use in the method of this invention include
methanol,
ethanol, 1-propanol, 2-propanol, tetrahydrofuran (THF), and acetonitrile, with
acetonitrile being most preferred.
Two compounds (in this case, DNA fragments of different size} can
only be separated if they have different partition coefficients (K). The
Nernst
partition coefficient is defined as the concentration of an analyte (A) in the
stationary phase divided by its concentration in the mobile phase:
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K = Lls
fAlm
The partition coefficient (K) and the retention factor (k) are related through
the
following equations:
K - nA Vm and k - nAs
the quotient VmNs is also called phase volume ratio (~). Therefore:
k - K~
To calculate the sorption enthalpies, the following fundamental
thermodynamic equations are necessary:
In K= - OG , In k= - OGso,~ + In cl~ and ~Gso,P = OHso,p - TOSso,P
RT RT
By transforming the last two equations, we obtain the Van't Hoff equation:
In k = - OH~,~ + ~Sso,~ + In ~
RT R
From a plot In k versus 1?, the sorption enthalpy OHso,P can be obtained from
the slope of the graph (if a straight line is obtained). OSso~p can be
calculated if
the phase volume ratio (~) is known.
Experiments on silica beads coated with poly{styrene-divinylbenzene)
also give a negative slope for a plot of In k versus 1/T, although the plot is
slightly curved.
If the acetonitrile is replaced with methanol, the retention factor k
decreases with increasing temperature, indicating the retention mechanism is
an exothermic process (OHso,P < 0).
The thermodynamic data (as shown in the Examples hereinbelow)
reflect the relative affinity of the DNA-counter ion agent complex for the
beads
of the inventipn and the elution solvent. An endothermic plot indicates a
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preference of the DNA complex for the bead. An exothermic plot indicates a
preference of the DNA complex for the solvent over the bead. The plots
shown herein are for alkylated and non-alkylated surfaces as described in the
Examples. Most liquid chromatographic separations show exothermic plots.
In addition to the beads themselves being substantially metal-free, we
have also found that, to achieve optimum peak separation during RPLPC, the
separation column and all process solutions held within the column or flowing
through the column should be substantially free of multivalent cation
contaminants (e.g. Fe(III), Cr(lll), and colloidal metal contaminants). As
described in commonly owned, copending U.S. application Ser. No.
08/748,376, (filed November 13, 1996), this can be achieved by supplying
and feeding solutions entering the separation column with components which
have process solution-contacting surfaces made of material which does not
release multivalent cations into the process solutions held within or flowing
through the column, in order to protect the column from multivalent cation
contamination. The process solution-contacting surfaces of the system
components are preferably material selected from the group consisting of
titanium, coated stainless steel, passivated stainless steel, and organic
polymer. Metals found in stainless steel, for example, do not harm the
separation, unless they are in an oxidized or colloidal partially oxidized
state.
For example, 316 stainless steel frits are acceptable in column hardware, but
surface oxidized stainless steel frits harm the DNA separation.
For additional protection, multivalent cations in eluent solutions and
sample solutions entering the column can be removed by contacting these
solutions with multivalent cation capture resin before the solutions enter the
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column to protect the resin bed from multivalent cation contamination. The
multivalent capture resin is preferably cation exchange resin and/or chelating
resin.
Other features of the invention will become apparent in the course of
the following descriptions of exemplary embodiments which are given for
illustration of the invention and are not intended to be limiting thereof.
All references cited herein are hereby incorporated by reference in
their entirety.
Procedures described in the past tense in the examples below have
been carried out in the laboratory. Procedures described in the present tense
have not been carried out in the laboratory, and are constructively reduced to
practice with the filing of this application.
EXAMPLE 1
C-18 Bonded Phase Standard Phase
To a 1000-mL round bottomed flask, add 200 g of nonporous, 2 pm
silica and one small stirring egg. Transfer flask with silica to an oven and
heat at 125°C overnight (i.e., at least 8 hours). Have heating mantle
and
condenser set up.
The C-18 bonding reagent, n-octadecyldimethylsilane, is a waxy white
solid to semi-solid at room temperature. To transfer, open the bottle in a
hood and gently warm with a heat gun (note: pressure can build up in stored
chlorosiiane bottles, and they should be handled as if they were HCI, as upon
contact with moisture, HCI is the side product).
To a second flask, transfer 125 g of the n-octadecylmethylchlorosilane
reagent, 10 mL of chloroform, 400 mL of toluene, and 65 mL of pyridine. Mix
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the liquid reagents by swirling, and then add to the dried silica and swirl
until
all of the silica is suspended. Attach the refluxing condenser and bring the
mixture to reflux for 15 hours. Let the mixture cool, such that refluxing has
stopped. Add the capping reagent package of 20 mL of trimethylchlorosilane,
6 mL of hexamethylsilane in 20 mL of toluene. Resuspend the mixture and
bring the system back to reflux for 6 hours. Let the mixture cool to room
temperature.
Transfer to a Buchner funnel and wash with three 200-mL aliquots of
methanol, followed by three 200-mL aliquots of acetone. Air dry for at least
0.5 hour, and then dry in the oven at 100°C overnight.
Submit sample for elemental analysis, and percent carbon.
Dried bonded phase is now ready for column packing.
EXAMPLE 2
CN Bonded Phase, Cyano Phase
To a 1000-mL round-bottomed flask, add 200 g of nonporous, 2 Nm
silica, one stirring egg, and place in an oven at 125°C overnight
(i.e., at least
8 hours) to dry. To the dried silica, add 100 rnL of the 3-
cyanopropylmethyldichlorosilane, 10 mL of chloroform, 450 mL of toluene,
and 50 mL of pyridine. Suspend the mixture and bring to reflux for 15 hours.
Cool filter and wash in a Buchner funnel with one 200-mL aliquot of toluene,
followed by two 200-mL aliquots of methanol. Transfer to a beaker and add
300 mL of 50:50 methanol:water, pH 5.5 with HCI. Suspend and let sit at
room temperature for 1 hour. Filter onto Buchner funnel and wash phase with
methanol and acetone. Transfer to the 1000-mL round-bottomed flask and
dry in oven overnight.
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Next, endcap by adding 20 mL of trimethylchlorosilane, 6 mL of
hexamethyl-disilane, 350 mL of toluene, 10 mL of chloroform, and 25 mL of
pyridine to the dried bonded phase, and bring to reflux for 6 hours. Cool the
resulting mixture, transfer to a Buchner funnel, and wash with three 200-mL
aliquots of methanol, followed by three 200-mL aliquots of acetone. Air dry
for at least 0.5 hour, and then dry in the oven at 100°C overnight.
Submit a sample for elemental analysis.
The bonded phase is now ready for column packing.
EXAMPLE 3
Diocfy! Silyl Phase - C-8X2
Repeat all of the steps for CN bonded phase, but replace 3-
cyanopropylmethyldichlorosilane with 100 mL of dioctyl dichlorosilane.
EXAMPLE 4
Acid Wash Treatment
The procedures of Example 1 are repeated but the silica is washed
with 500 mL of 100 mM HCI and then water prior to drying. The product is
washed with 500 mL of 100 mM HCI after cooling and prior to the methanol
wash.
EXAMPLE 5
The product of Example 1 is coated with 100 mL of dichloromethane
containing 1 gram of divinylbenzene and 10 mg of benzoylperoxide. The
dichloromethane is removed by rotary evaporation until the monomer is
coated onto the beads. While rotating very slowly, the temperature is
increased to 70°C for 8 hours. The product is washed with methanol.
This procedure is repeated with the product of Example 4.
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EXAMPLE 6
The procedure of Example 5 is repeated with stearyidivinyl benzene in
place of divinylbenzene. This procedure is repeated with the product of
Example 4.
EXAMPLE 7
Fifteen (15) grams of the nonporous silica particles, 50 mL of 2,2,4-
trimethylpentane, and 25 mL of vinyltrichlorosilane are refluxed for 2 hours.
The modified silica is then washed several times with both 2,2,4-
trimethylpentane and acetone and dried at 80°C.
Five (5) grams of the vinyl-coated silica particles prepared as
described above are placed in a round bottom flask. Twenty-five mL of
acetonitrile containing 2 g of a vinyl monomer (divinylbenzene, styrene,
acrylonitrile, acrylic acid, butyl methacrylate, or 2-hydroxy methacrylate)
are
added and the mixture well dispersed. Twenty-five mL of acetonitrile
containing 0.2 g of dibenzoyl peroxide is added, and the mixture is refiuxed
for 2 hours.
The products are extracted with acetonitrile and then acetone to
remove unreacted monomers and oligomers from the particle.
1n the case of the acrylic acid-modified silica, extractions with water are
also carried out.
The packing materials are dried at 80°C prior to packing.
EXAMPLE 8
Standard Procedure for Testing the Performance of Separation Media
Separation particles are packed in an HPLC column and tested for
their ability to separate a standard DNA mixture. The standard mixture is a
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pUCl8 DNA-Haelll digest (Sigma-Aldrich, D6293) which contains 11
fragments having 11, 18, 80, 102, 174, 257, 267, 298, 434, 458, and 587
base pairs, respectively. The standard is diluted with water and five NL,
containing a total mass of DNA of 0.25 ~.g, is injected.
Depending on the packing volume and packing polarity, the procedure
requires selection of the driving solvent concentration, pH, and temperature.
The separation conditions are adjusted so that the retention time of the 257,
267 peaks is about 6 to 10 minutes. Any one of the following solvents can be
used: methanol, ethanol, 2-propanol, 1-propanol, tetrahydrofuran (THF), or
acetonitrile. A counter ion agent is selected from trialkylamine acetate,
trialkylamine carbonate, trialkylamine phosphate, or any other type of cation
that can form a matched ion with the polynucleotide anion.
As an example of this procedure, FIG. 4 shows the high resolution of
the standard DNA mixture using octadecyl modified, nonporous
poly(ethylvinylbenzene-divinylbenzene) beads. The separation was
conducted under the following conditions: Eluent A: 0.1 M TEAA, pH 7.0;
Eluent B: 0.1 M TEAA, 25% acetonitrile; Gradient:
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Time %A %B
min
0.0 65 35
3.0 45 55
10.0 35 65
13.0 35 65
14.0 0 100
15.5 0 100
16.5 65 35
The flow rate was 0.75 mUmin, detection UV at 260 nm, column temp.
50°C. The pH was 7Ø
As another example of this procedure using the same separation
conditions as in FIG. 4, FIG. 5 is a high resolution separation of the
standard
DNA mixture on a column containing nonporous 2.1 micron beads of
underivatized polystyrene-divinylbenzene).
EXAMPLE 9
This example demonstrates the high resolution separation of DNA
restriction fragments using octadecyl modified, nonporous silica reverse
phase material, as described in Example 1. The experiment is conducted
under the following conditions: Column: 50x4.6 mm i.d. Mobile phase: 0.1
M TEAA, pH 7Ø Gradient: 8.75 - 11.25% acetonitrile in 2 minutes, followed
by 11.25 - 14.25% acetonitrile in 10 minutes, 14.5 - 15.25% acetonitrile in 4
minutes, and by 15.25 - 16.25% acetonitrile in 4 minutes. Flow rate 1
mUmin. Column temperature: 50°C. Detection: UV at 254 nm. Sample:
Mixture of 0.75 Ng pBR322 DNA-Haelll restriction digest and 0.65 Ng x174
DNA-Hinc II restriction digest.
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A high resolution separation is obtained by optimizing the
concentration of triethylammonium acetate (TEAA), shape of the gradient
curve, column temperature, and flow rate. The resolution of peaks is
continuously enhanced in going from 25 mM to at least 7 25 mM of TEAA.
S The gradient is optimized by decreasing the steepness of the gradient curve
with increasing fragment lengths of DNA molecules. The best separations of
double-stranded DNA molecules are accomplished at about 30°C to
50°C.
Denaturation of DNA at higher than about 50°C prevents utilization of
higher
column temperatures for double-stranded DNA fragments, although single-
stranded DNA separations can be performed at temperatures up to 80°C
and
higher.
EXAMPLE 10
If the gradient delay volume is minimized, the separation of PCR
products and hybrid DNA derived from various sources of DNA, including
living and dead organisms (animal and plant), as well as parts of such
organisms (e.g., blood cells, biopsies, sperm, etc.) on octadecyl modified,
nonporous poly-(ethylvinylbenzene-divinylbenzene) coated beads can be
achieved with run times under 2 minutes.
The analysis of PCR products and hybrid DNA usually requires only
separation and detection of one or two species of known length. Because of
this, the resolution requirements are considerably less severe than for
separations of DNA restriction fragments. Such less stringent resolution
requirements allow the utilization of steep gradients and, consequently, lead
to still shorter run times. The recovery rate for a DNA fragment containing
404 base pairs is about 97.5%.
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Unlike capillary electrophoresis (CE), PCR samples do not have to be
desalted prior to analysis by M1PC. This represents a decisive advantage of
MIPC over CE. With MIPC, it is thus possible to achieve a fully automated
analysis of PCR samples if an automatic autosampier is utilized. Moreover,
since the volume of sample injection is known, in contrast to CE, quantitation
over several orders of magnitude can be achieved without the need for an
internal standard, hence allowing the quantitation of gene expression, as well
as the determination of virus titers in tissues and body fluids. A fully
automated version of the method of the invention can be used to discriminate
(distinguish) normal from mutated genes, as well as to detect oncogenes,
bacterial and viral genome polynucleotides (hepatitis C virus, HIV,
tuberculosis) for diagnostic purposes. Moreover, adjustment of column
temperature allows one to moderate the stringency of hybridization reactions
or to separate heteroduplex from homoduplex DNA species.
The suitability of the polymer-coated beads of the invention for clinical
use is described under the following conditions: Column: 50x4.6 mm i.d.
Mobile phase: 0.1 M TEAR, pH 7Ø Gradient: 11.25 - 13.75% acetonitrile in
1 minute, followed by 22.5% acetonitrile for 6 seconds, and 11.25%
acetonitrile for 54 seconds. Flow rate: 3 mUmin. Column temperature:
50°C. Detection: UV at 256 nm. Sample: 20 NI of a PCR sample. In the
separation, the following elution order is obtained: 1=unspecific PCR product,
2=PCR product having 120 base pairs, 3=PCR product having 132 base
pairs, and 4=PCR product having 167 base pairs.
PCR methods and processes are described by R. K. Saiki et al. in
Science, 23):1350-1354 (1985) and K. B. Mullis in U.S. Patent No. 4,863,202.
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These references are incorporated herein by reference for a more complete
description of methods and processes for obtaining PCR samples which can
be separated using the method of the present invention.
The repetitive analysis of PCR products using the method of the
invention is highly reproducible under the described analytical conditions.
The results are not in any way influenced by the preceding injection. The
present method is highly suitable for routine use under real conditions in
clinical laboratories.
EXAMPLE 11
The following describes a separation of single-stranded DNA. A siiica-
C18 column, as described in Example 1, 1.5 micron, 30x4.6 mm i.d., is used
with a linear gradient of 2.5 - 12.5% acetonitrile in 0.1 M triethylammonium
acetate in 40 minutes at 1 mUmin and 40°C. A mixture of p(dC)12-18 and
p(dT)12-18 oligonucleotides is separated; with the first mixture eluting
between 5 and 15 minutes, and the second mixture eluting between 15 and
30 minutes.
EXAMPLE 12
Sorption Enthalpy Measurements
Four fragments (174 base pair, 257 base pair, 267 base pair, and 298
base pair, found in 5 NI pUCl8 DNA-Haelll digest, 0.04 Ng DNA/NI) of a DNA
digest are separated under isocratical conditions at different temperatures
using C-18 alkylated polystyrene-divinyibenzene) polymer beads. Conditions
used for the separation are: Eluent: 0.1 M triethylammonium acetate,
14.25% (v/v) acetonitrile at 0.75 mUmin, detection at 250 nm UV,
temperatures at 35, 40, 45, 50, 55, and 60°C, respectively. A plot of
In k
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versus 1/T (FIG. 6) shows that the retention factor k is increasing with
increasing temperature. This indicates that the retention mechanism is based
on an endothermic process (OHso,P > 0).
The same experiments on non-alkylated polystyrene-divinylbenzene)
beads (FIG. 7) give a negative slope for a plot of In kversus 1/T, although
the
plot is slightly curved.
The same experiments on alkytated polystyrene-divinytbenzene)
beads but the acetonitrile solvent is substituted with methanol (FIG. 8) gives
a
plot In kversus 1!l'shows the retention factor k is decreasing with increasing
temperature. This indicates the retention mechanism is based on an
exothermic process (OHso,P < ~). Replacing the alkylated and non-alkylated
polymer beads with silica beads having a coating of alkylated poly(styrene-
divinylbenzene) and non-alkylated alkylated polystyrene-divinylbenzene) will
give the same results.
EXAMPLE 13
Separations with alkylated polystyrene-divinylbenzene) beads
Etuents are chosen to match the desorption ability of the elution
solvent to the attraction properties of the bead to the DNA-counter ion
complex. As the polarity of the bead decreases, a stronger (more organic) or
higher concentration of solvent will be required. Weaker organic solvents
such as methanol are generally required at higher concentrations than
stronger organic solvents such as acetonitrile.
FIG. 9 shows the high resolution separation of DNA restriction
fragments using octadecyl modified, nonporous poly(ethytvinylbenzene-
divinylbenzene) beads. The experiment was conducted under the following
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conditions. Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M tetraethylacetic
acid (TEAA), pH 7.2; gradient: 33-55% acetonitrile in 3 min, 55-66%
acetonitrile in 7 min, 65% acetonitrile for 2.5 min; 65-100% acetonitrile in 1
min; and 100-35% acetonitrile in 1.5 min. The flow rate was 0.75 mUmin,
detection UV at 260 nm, column temp. 51 °C. The sample was 5 p.l (= 0.2
p,g
pUCl8 Hea III digest).
Repeating the above procedure replacing the acetonitrile with 50.0%
methanol in 0.1 M TEAA gives the separation shown in FIG. 10.
Repeating the above procedure replacing the acetonitrile with 25.0%
ethanol in 0.1 M TEAA gives the separation shown in FIG. 11.
Repeating the above procedure replacing the acetonitrile with 25%
vodka (Stolichnaya, 100 proof) in 0.1 M TEAA gives the separation shown in
FIG. 12.
The separation shown in FIG. 13 was obtained using octadecyl
modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as
follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;
gradient: 12-18% 0.1 M TEAA and 25.0% 1-propanol (Eluent B) in 3 min, 18-
22% B in 7 min, 22% B for 2.5 min; 22-100% B in 1 min; and 100-12% B in
1.5 min. The flow rate was 0.75 mUmin, detection UV at 260 nm, and column
temp. 51 °C. The sample was 5 p,l (= 0.2 p.g pUCl8 DNA-Haelll digest).
The separation shown in FIG. 14 was obtained using octadecyl
modified, nonporous poly(ethyivinylbenzene-divinylbenzene) beads as
follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;
gradient: 15-18% 0.1 M TEAA and 25.0% 1-propanol (Eluent B) in 2 min, 18-
21 % B in 8 min, 21 % B for 2.5 min; 21-100% B in 1 min; and 100-15% B in
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1.5 min. The flow rate was 0.75 mUmin, detection UV at 260 nm, and column
temp. 51°C. The sample was 5 p.l (= 0.2 ~.g pUCl8 DNA-Haelll digest).
The separation shown in FIG. 15 was obtained using octadecyl
modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as
follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;
gradient: 35-55% 0.1 M TEAA and 10.0% 2-propanol (Eluent B) in 3 min, 55-
65 % B in 10 min, 65% B for 2.5 min; 65-100% B in 1 min; and 100-35% B in
1.5 min. The flow rate was 0.75 mUmin, detection UV at 260 nm, and column
temp. 51 °C. The sample was 5 ~.I (= 0.2 p,g pUC18 DNA-Haelll digest).
The separation shown in FIG. 16 was obtained using octadecyf
modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as
follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.05 M TEA2HP04, pH 7.3;
gradient: 35-55% 0.05 M TEA2HPO,and 10.0% 2-propanol (Eluent B) in 3
min, 55-65% B in 7 min, 65% B for 2.5 min; 65-100% B in 1 min; and 100-
65% B in 1.5 min. The flow rate was 0.75 mUmin, detection UV at 260 nm,
and column temp. 51°C. The sample was 5 ~.I (= 0.2 p.g pUCl8 DNA-Haelll
digest).
The separation shown in FIG. 17 was obtained using octadecyl
modified, nonporous poly(ethylvinylbenzene-divinylbenzene} beads as
follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;
gradient: 6-9% 0.1 M TEAA and 25.0% THF (Eluent B) in 3 min, 9-11 % B in
7 min, 11 % B for 2.5 min; 11-100% B in 1 min; and 100-6% B in 1.5 min. The
flow rate was 0.75 mUmin, detection UV at 260 nm, and column temp. 51
°C.
The sample was 5 p,l (= 0.2 pg pUCl8 DNA-Haelll digest).
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EXAMPLE 14
Isocraticlgradient separation of dsDNA
The following is an isocratic/gradient separation of dsDNA on a
polystyrene coated silica base material. Isocratic separations have not been
performed in DNA separations because of the large differences in the
selectivity of DNA/alkylammonium ion pair for beads. However, by using a
combination of gradient and isocratic elution conditions, the resolving power
of a system can be enhanced for a particular size range of DNA. For
example, the range of 250-300 base pairs can be targeted by using an eluent
of 0.1 M TEAA, and 14.25% acetonitrife at 0.75 mUmin at 40°C on 50 x
4.6
mm crosslinked polystyrene coated silica reverse phase column, 2.0 micron.
The pUCl8 DNA-Haelll digest was injected under isocratic conditions and
257, 267 and 298 base pairs DNA eluted completely resolved. Then the
column was cleaned from larger fragments with 0.1 M TEAA/25% acetonitrile
at 9 minutes. FIG. 18 shows a separation using the same elution conditions
but performed on a polystyrene-divinylbenzene) polymer based column. In
other examples, there might be an initial isocratic step (to condition the
column), then a gradient step (to remove or target the first group of DNA at a
particular size), then an isocratic step (to separate the target material of a
different size range) and finally a gradient step to clean the column.
While the foregoing has presented specific embodiments of the
present invention, it is to be understood that these embodiments have been
presented by way of example only. It is expected that others will perceive
and practice variations which, though differing from the foregoing, do not
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depart from the spirit and scope of the invention as described and claimed
herein.
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