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BACKGROUND OF THE INVENTION
The present invention relates to packing materials for liquid
chromatographic or catalytic columns, and more particularly it relates to
an improved packing material for use in high performance liquid
chromatography and to a method for making and using such a packing
material.
Liquid column (LC) packing materials are usually porous materials which
possess adsorptive or catalytic sites on the pore walls. They may be used
as packings in columns or as loose material in vessels. LC packing
materials typically are porous particles. However, they may instead be
fibers or membranes. Porous membranes also provide filtration. When the
pore size of the adsorptive or catalytic membrane excludes large proteins,
then ultrafiltration is combined with adsorption or catalysis. Membranes
may have small pores throughout their mass which exclude protein.
Alternatively, the membrane may be a composite of sintered or adhered
porous particles, in which case the pores between particles are large
whereas the pores within the sintered or adhered particles are small. For
example, Kontes Glass Co. markets a thick porous membrane of polyvinyl
chloride upon whose large pore walls are attached small porous silica
particles. The pores of the silica particles are much smaller than the
pores upon whose walls the silica particles are attached.
Liquid column chromatographic techniques are used for the separation,
analysis, and purification of small molecules as well as of polymers such
as proteins in solution. Such separations are mediated either by surface
interactions or by size or electrostatic exclusion interactions.
Surface-mediated separations require a degree of adsorption of solute to
the packing surface. The adsorption is usually due to physisorption and
can be driven by hydrophobicity for the case of lipophilic solutes
(reverse phase or hydrophobic interaction chromatography), by ion exchange
for the case of charged solutes, and by bioaffinity interactions. Often
several mechanisms occur simultaneously though one usually dominates. The
adsorption can also be due to covalent bond formation to the support. For
example, dissolved saccharides can form a covalent boronate bond to
phenylboronic acid immobilized on a support.
Analysis and purification of biological fluids represents a particularly
important application of liquid chromatography. Such biofluids include
blood, plasma, serum, urine, tissue extracts and fermentation and cell
cultures. Such biological fluids are generally highly aqueous with minimal
organic cosolvent content. It is often advantageous to maintain a highly
aqueous state since addition of organic cosolvents reduces the solubility
of many such proteins, and hence can cause precipitation and loss of some
components prior to chromatography. In other instances, precipitation is
used to remove interfering substances.
Virtually all proteins are strongly adsorbed by reverse phase packings when
the mobile phase in the column is weakly eluting, as is the case with
highly aqueous mobile phases. Salting out effects can accentuate such
adsorption. All proteins are also adsorbed by ion exchange packings when
their charge is counter to that of the ion exchange sites on the packing
surface. This condition can be attained by adjusting the mobile phase pH
to the appropriate side of the isoelectric point (IEP). Proteins are also
known to be adsorbed by unbonded chromatographic silica.
Size exclusion chromatography requires minimal adsorption to the packing
surface. Such surfaces are typically very hydrophilic when proteins are
subjected to size exclusion chromatography using highly aqueous mobile
phases containing little or no organic cosolvent.
Liquid chromatography using a reverse phase packing has been found to be an
effective tool in both qualitative and quantitative analysis for drug
substances in blood, serum, plasma or urine. Typically the reverse phase
packing material is made up of bonded alkyl silica and most typically the
packing is a porous silica having octadecylsilane (ODS) bonded to it.
Although the efficiency of such packing materials is good, they have a
limited life. While ODS packings absorb the lipophilic drug substances
from the sample, they also absorb proteinaceous substances which tend to
interfere with fractionation of the drug substance from other materials
contained in the sample. This eventually leads to a complete fouling of
the chromatographic column. Therefore, it has previously been necessary to
carry out a preliminary sample preparation procedure to remove the
troublesome proteins.
In the most conventional way, the proteins are precipitated, the aqueous
supernatant is extracted with a water-immiscible organic solvent, the
organic solvent is removed from the extract by evaporation, and the
analyte residue is reconstituted in mobile phase before analysis by
high-pressure liquid chromatography. This method is very time-consuming
and cost-inefficient.
A second method currently employed involves the adsorption of analytes onto
a reverse phase packing of octadecylsilane bonded to silica in a small
disposable column. Although this technique can be automated, the columns
can be used for only one sample because proteins remain on the packing,
and as a result the technique is also cost-inefficient for multiple
samples.
In a third method, a reverse phase packing of octadecylsilane bonded to
silica is introduced into a precolumn, which is separated from, but
connectable to, an analytical column by a switching valve arrangement.
Serum samples are injected directly into the precolumn, where the proteins
are denatured and accumulated, and the deproteinated analyte solution is
passed into the analytical column for fractionation. After approximately
three injections, the precolumn must be backflushed to remove the protein
residue. This interruptive backflush is time-inefficient for a large
number of samples. Furthermore, the octadecylsilane packing eventually
deteriorates because proteins cannot be completely removed therefrom.
Accordingly, for reverse phase liquid chromatography it would be desirable
to have a packing material which is less protein adsorptive. In my U.S.
Pat. Nos. 4,773,994, 4,778,600, 4,782,040, 4,950,634 and 4,950,635 there
are disclosed improved reverse phase packing materials, termed dual zone
materials. The dual zone reverse phase packing materials display a reduced
degree of serum protein adsorption due to a lipophobic fluoroalkyl phase
in the external zone. The pore size of the packing material is small so
that size exclusion prevents the protein from reaching the internal zone
where the lipophilic partitioning phase retains and separates drug
substances. Although the lipophobic phase reduces protein adsorption when
the mobile phase contains greater than or equal to 20 percent organic
cosolvent, further minimization of protein adsorption would be desirable,
especially when using more highly aqueous mobile phases in the column.
Other approaches to achieving a packing which has an exterior
non-adsorptive to proteins combined with a size-excluded reverse phase
interior are known. Size-excluded enzymes have been used to selectively
modify the exterior of silica bearing covalently bonded oligopeptides.
See, e.g., I. H. Hagestam et al, "Internal Surface Reverse Phase Silica .
. . ", J. Chrom. 351, (1986) p. 239. However, the scope of choices for the
internal partitioning phase is severally constrained since many desirable
partitioning phases may not be easily embodied in an oligopeptide while
still remaining cleavable by an enzyme.
An approach that eliminates most constraints on the internal partitioning
phase is to coat the packing with sufficient protein to prevent further
protein adsorption. When large amounts of serum albumin or plasma are
loaded onto an ODS-silica column, the column adsorbs no further protein
and is said to be saturated. The silica is selected to have a pore size
that excludes the protein from the pores so that the internal reverse
phase remains unfouled and separatively active towards small lipophilic
solutes such as drugs in plasma. However, the coating is removed by
strongly eluting mobile phases. Hence the column saturation is lost during
periodic column cleanup or during gradient elution chromatography.
Most of the coating can be permanently attached by passing 100% methanol
through the column to denature and physically crosslink the coating.
However, some saturation is lost after applying this crosslinking method,
so that the entire treatment must be performed several times. After
several cycles of saturation followed by denaturation, a permanently
saturated column results. Such columns have been used to directly inject
plasma and serum samples for LC analysis of drugs. See, e.g., H. Yoshida
et al, "Some Characteristics of a Protein-Coated ODS Column . . . ",
Chromatographia, Vol. 19, 1985, pp. 466-472.
These columns also have significant disadvantages. The Height Equivalent
Theoretical Plate height rises more than 75 micrometers after this
treatment. Even for the relatively inefficient 26 micrometer ODS-silica
used, the plate height rise caused an efficiency loss of over 70%. Smaller
silica particles would display much greater efficiency loss if the same
plate height rise occurred, as expected if the rise was due to the
diffusional barrier of the coating. However, the cause of this relatively
low efficiency has not been proven in the literature.
Simple calculus shows that the volume fraction due to the shell of coating
relative to the entire coated packing is given by 6 t/D, where t is the
coating thickness and D is the silica particle diameter in micrometers.
Hence, the coating thickness is given by W*D/(600.alpha.), where W is the
protein weight percent and .alpha. is the ratio of the coating volumetric
density to that of the support.
Although not reported in the literature, the amount of protein in the
saturated solvent-stable column of Yoshida was found to be very high when
compared to the support particle diameter. The product of weight percent
times diameter was 2.5.times.26=65. Given that the bulk densities of
protein and of porous silica are about equal, the data could suggest that
a thick coating formed on the order of 1100 angstrom thick. Since a single
albumin molecule is approximately an 80 angstrom diameter sphere, a
multilayer coating may have resulted. Thick coatings are known to degrade
efficiency by creating a large barrier to solute diffusion. See, e.g.,
Kirkland, J., "High Speed Liquid-Partition Chromatography With Chemically
Bonded Organic Stationary Phase", Journal of Chromatographic Science, Vol.
9 (1971) pp. 206-214. Thus it appears that there is a need to obtain a
permanently saturated but thin protein coating on supports.
A second approach to imparting a crosslinked protein coating onto packing
materials employs simultaneous contact of glutaraldehyde with a
concentrated solution of protein in an unbonded silica slurry in water.
Such coated supports have high immobilized protein context and are useful
for chromatography of dissolved protein. The object of this approach is to
maximize the amount of immobilized protein short of creating an
impermeable composite through which liquid could not readily flow. In this
approach, the weak adsorption properties of the immobilized protein in the
packing material are useful. See, e.g., M. Tsuboi et al, "Chromatography
Carrier", Japanese Patent Application No. 198,334/85, Sept. 7, 1985. A
similar method uses a two-stage glutaraldehyde crosslinking procedure in
which the crosslinking was interrupted after a period of time by washing
away serum albumin that had not yet deposited on the silica. Subsequently,
more glutaraldehyde was added to ensure that the remaining albumin was
tightly crosslinked and permanently attached to the silica. The two stage
process ensured that large clumps of support particles were not glued
together. Such clumps disrupt flow through the column and degrade
efficiency. See, e.g., R. A. Thompson et al, "Sorbents Obtained by
Entrapment of Crosslinked Bovine Serum Albumin in Silica", Journal
Chromatography, Vol. 465 (1989) pp. 263-270.
The two-stage crosslinking approach resulted in chiral packing materials
useful for separating racemic mixtures. However, the efficiency for the
isomers of benzoin was only 10,000 plates/meter (P/M). The expected
efficiency for the 7 micrometer silica used is 30,000-40,000 P/M. The
protein weight percent was 13% and 21% for silica whose pore diameters
were 50 and 100 angstroms, respectively. The value of W*D of 119 could
suggest that a very thick coating formed, which is consistent with the
degraded column efficiency. Hence this approach is not favorable to
attaining a saturated but efficient coated packing material.
Yet another approach to forming a protein coating is to use glutaraldehyde
as a coupling agent in a first step by bonding it to an
aminopropyl-silica, leaving an immobilized aldehyde residue to which in a
second step protein can be bonded through the amino side chain of lysine
amino acid residues. Often sodium cyanoborohydride or pyridine borane is
used to stabilize the bond to the packing by reducing the intermediate
imine to the secondary amine. It is common in a final step to block
residual immobilized aldehyde by addition of an excess of some hydrophilic
primary amine such as tris(hydroxymethyl)aminomethane, glycine, or
ethanolamine to avoid non-specific bonding by aldehyde during affinity
chromatography. See, e.g., F. R. Bernath et-al, "Methods of Enzyme
Immobilization", in Manual of Industrial Microbiology and Biotechnology,
ed. A. L. Deman & N. A. Solomon, publ. Amer. Soc. Microbiology, Wash. D.C.
(1986) pp. 244-5. This approach immobilizes protein by forming covalent
bonds between it and the support. Although this approach yields usable
products, the partitioning phase is limited in scope since it must also
bear amino groups.
Column packing materials bearing biocatalytic residues are also subject to
fouling by the sample or process fluid. Such fouling can be due to
particulates or to large proteins and colloids in the process fluid.
Fouling by particulates physically blocks the column or membrane. However,
such fouling can be countered by backflushing. In contrast, fouling by
proteins is difficult to reverse, particularly when the protein adsorbs to
the packing exterior and obstructs the mouths of the pores. Consequently,
obstruction of diffusion of solute to the catalytic interior reduces the
activity. See, e.g., P. S. J. Cheetham, "Principles of Industrial
Enzymology", Handbook of Enzyme Technology, ed. A. Wiseman, publ. J. Wiley
(NY, 1985) pp 126-128. Reduction of protein adsorption to the packing
material would alleviate problems due to this source of fouling.
Thus, despite all of the recent advances in HPLC packing materials, the
need still exists for improved minimization of protein adsorption while
possessing an internal adsorptive or catalytic phase so as to improve
utility and extend the usefulness of such packing materials.
SUMMARY OF THE INVENTION
The present invention meets that need by providing a packing material for
liquid chromatographic or catalytic columns which is resistant to protein
adsorption. The packing material of the present invention is a porous
protein-adsorptive support having a coating of crosslinked protein on the
external surfaces thereof. "Crosslinked" refers to bonds formed between
protein molecules, although bonds solely within a given protein molecule
may be formed concomitantly. Surprisingly, it has been found that by
applying a relatively thin but saturated coating of physisorbed protein
and crosslinking in situ without displacing part of the coating, the
resultant packing material has a valuable combination of two properties:
permanently improved resistance to further protein adsorption yet minimal
thickness and hence minimal adverse effects that thick coatings generate.
Essentially it would appear that by controlling the degree of "protein
fouling" in this manner further fouling is minimized and the usefulness of
the packing material extended.
Reverse phase, cation exchange, and anion exchange organic resin or
silica-based supports are sufficiently protein-adsorptive to be used, as
is untreated silica. The preferred porous protein-adsorptive support is a
porous silica support such as porous silica having a pore diameter of 30
to 300.ANG., and a particle size of 1 to 500 micrometers; although, any
protein-adsorptive support may be used. When a porous silica support is
used it may be one already having an alkylsilane bonded to the surfaces
thereof or it may be a dual zone or mixed phase material such as that
shown in my U.S. Pat. No. 4,773,994, 4,778,600, 4,782,040, 4,950,634 and
4,950,635. In all instances, the coating of crosslinked protein covers the
external surfaces of the porous silica support overcoating the alkyl or
ketal-blocked-diol or fluoroalkyl or other external phase of the dual zone
or mixed phase material.
The protein should be of a sufficient molecular size as to be size-excluded
from the internal surfaces of the porous protein-adsorptive support.
Larger proteins may be made from smaller ones prior to adsorption to the
support. Such larger proteins will be size-excluded from a larger
pore-diameter support. Such solution phase crosslinking to make a larger
protein is well known in the field of biochemistry. Preferably the protein
is one having a molecular size of 40 to 400.ANG.. In this manner the
extent of coating of protein on the porous support is limited to the
external surfaces. Preferably, the protein is a water soluble one selected
from the group consisting of serum albumin, bovine serum albumin (BSA),
egg conalbumin, ovalbumin, and serum .alpha.-glycoprotein.
The process of applying the protein should be one that coats an amount of
protein to the support which is self-regulated by saturative adsorption to
be the minimal effective amount needed to prevent further protein
adsorption. Preferably the coating of crosslinked protein is applied to
the porous protein-adsorptive support at least in three steps, including
these: first, the porous support is contacted with an aqueous solution
(into which the protein has been dissolved) for a sufficient period of
time to form, and under conditions which form, a saturated coating of
physisorbed protein on the external surfaces of the porous support.
Second, the excess protein still in solution is washed away. Then, as a
third step, all of the protein is crosslinked in situ to form a coating of
crosslinked protein on the external surfaces of the porous support. Any
conventional crosslinking reaction may be used, including those which use
heat, or oxidizing agents or chemical crosslinking agents. The use of
chemical crosslinking agents is preferred. Most preferred is the use of
crosslinking agents selected from the group consisting of glutaraldehyde,
formaldehyde, N, N' dimethylaminopropyl ethyl-carbodiimide, and bis
(sulfosuccinimidyl) suberate.
Preferred preparation of the coating when the support is a membrane is by
immersion in a stirred liquid so that the support may be subjected to a
series of operations by sequentially changing the liquid: Be wetted by
solvent or solvents be coated by deposition of protein, be washed to
remove excess protein in solution, and be brought into contact with
crosslinking agents.
The amount of protein coating needed to saturate a particulate
protein-adsorptive support depends on the particle diameter of the support
and is independent of pore diameter if the protein is large enough, i.e.,
of the size indicated above. Generally, a coating of solvent-stable
protein between 2.0/D and 10.0/D weight percent of the packing material
whose particle diameter is D micrometers may be used. Preferred is
preparation of the coating where the support is confined in a tube so that
the support may be subjected to a sequence of operations: Be wetted by
solvent or solvents, receive protein deposition, be washed to remove
excess protein in solution, and be brought into contact with crosslinking
agents by simply displacing each liquid that coresides with the support in
the tube by the next liquid. Most preferred is preparation of a coating
where the particulate support is coated in a slurry whose liquid
composition is changed in a sequence of steps to carry out the above
operations. A preliminary priming step may be used for coatings made using
the slurry approach when the support is hydrophobic, and thus not
dispersible in an aqueous solvent containing no surfactant or organic
cosolvent.
In the preferred method, porous silica having pore diameters of 30 to
300.ANG. and particle size of 1 to 500 micrometers in a column or in a
slurry containing at least 1 weight percent of support per weight of
liquid is contacted with an aqueous solution containing at least 10/D
parts of protein to 100 parts of support by weight where D is the support
particle diameter in micrometers, and having a pH within 0.5 of the IEP of
the protein at 4.degree. C. to 70.degree. C. for a period of 5 minutes to
24 hours with vigorous contact of liquid with support. These deposition
conditions represent those in which the maximum amount of protein
adsorption would occur in actual use. The above-mentioned preferred
proteins will under these conditions form a saturated coating. The protein
coating is then washed to remove excess protein still in solution. During
the third stage of the process, the coating is crosslinked with
conventional crosslinking agents such as those mentioned above under
conditions which will not displace the physisorbed protein coating. In the
preferred embodiment, those conditions are use of glutaraldehyde at a
temperature of 4.degree. C. to 70.degree. C. for a time of 30 minutes to
24 hours.
The resultant packing material has a solvent-stable coating on the external
surfaces that resists further adsorption by many different proteins, while
continuing to provide the adsorptive or catalytic properties of the groups
on the internal surfaces for separation, analysis, or alteration to small
molecules. Surprisingly, the packing materials of the present invention
perform better as HPLC packings in the sense that the coating of
crosslinked protein had less adverse impact on the chromatography and had
equal or better resistance to further protein adsorption than previously
known protein-resistant packing materials.
Thus the packing materials of the present invention are particularly useful
in a method of analysis by liquid chromatography in which an analyte
containing biological fluid to be analyzed is contacted with a liquid
chromatography column containing the packing material as the present
invention. For example, it has been found that bovine serum albumin
crosslinked onto reverse phase HPLC columns using the method of the
present invention gave greatly improved gradient HPLC of drugs in directly
injected serum samples. Packings for use in HPLC isocratic, gradient,
column-switching, and SPE columns or as loose batch adsorbents for
pharmaceutical analyses and purifications can also be prepared by the
method of the present invention.
Accordingly, it is an object of the present invention to provide a packing
material having improved liquid chromatography characteristics, to provide
a method for making such packing materials. Other objects and advantages
of the invention will become apparent from the following detailed
description of the appended claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The porous support for the packing materials of the present invention may
be any porous solid which is mildly hydrophobic and/or protein absorptive,
including ion exchange packings and even some bioaffinity packings.
However, preferred is a porous protein-adsorptive support having hydroxyl
groups on its surface such as porous metalloid oxides, porous metallic
oxides, and porous mixed metallic oxides. Such materials include silica,
silica gel, alumina, stannia, titania, zirconia, and the like. However,
HPLC packings are almost always silica particles or silica gels and
therefore porous silica is the most preferred. Accordingly, the porous
protein-adsorptive support will hereinafter be referred to as a porous
silica support. Preferably the pore diameter is 30 to 300 angstroms and
most preferably around 60 angstroms. Preferably the particle size is 1 to
500 micrometers and most preferably is 3 to 60 micro-meters.
Prior to treatment in accordance with the present invention the porous
silica support may undergo a silylation process with, for example, an
octadecylsilane as is well known. Alternatively, the porous silica support
may be converted into a dual zone or mixed phase material as is taught in
my U.S. Pat. Nos. 4,773,994, 4,778,600, 4,782,040, 4,950,634 and
4,950,635, the disclosures of which are hereby incorporated by reference.
In all instances, the coating of crosslinked protein covers the external
surfaces of the porous silica support overcoating the alkyl or
ketal-blocked-diol or fluoroalkyl or other external phase of the dual zone
or mixed phase material.
A preliminary priming step is preferred for coatings made using the slurry
approach when the support is hydrophobic, and thus not dispersible in an
aqueous solvent containing no surfactant or organic cosolvent. During the
priming step, a subsaturative amount of protein is deposited on the
support while the support is suspended in a mixture of water and
sufficient organic cosolvent to wet and disperse the support prior to its
exposure to protein. A sufficient amount of water-miscible organic
cosolvent such as methanol, ethanol, propanol, acetonitrile,
tetrahydrofuran (THF), acetone etc. is used ranging between 30 and 70
volume percent, depending on the degree of hydrophobicity of the support
and on the particular cosolvent. Most preferably, the aqueous portion of
the mixed solvent is buffered water whose pH is far from the protein's IEP
to enhance protein solubility in the solvent mixture. The amount of
protein added should dissolve in the solvent mixture. Preferably the
protein may be added to the slurry as a predissolved, concentrated
solution in water or buffered water or solvent mixture. Optionally, the pH
of the protein-containing slurry may then be gradually adjusted to reduce
protein solubility and thus increase the efficiency of utilization of the
protein by increasing the fraction which is deposited onto the support.
Solubility is near a minimum when the pH of the solvent mixture is equal
to that obtained when the initial pH of the buffered water is near the IEP
of the protein, as is well known. See, for example, R. Scopes, Protein
Purification, Springer-Verlag, N.Y. (1986) pp. 52-60. Under the above
constraints, protein will not precipitate as separate particles which
would be difficult to remove from the support particles.
In any event, as a first actual treatment step in the present method,
either an untreated porous silica support or a prior-treated porous silica
support, is contacted with an aqueous solution into which a protein has
been dissolved. As mentioned, the protein is selected relative to the pore
diameter of the porous silica so as to be size-excluded from the internal
surfaces of the porous silica. Thus, it preferably has a molecular size of
40 to 400.ANG.. It should also be water soluble. The preferred proteins
are serum albumin, bovine serum albumin (BSA), egg conalbumin, ovalbumin,
and serum .alpha.-glycoprotein.
The amount of protein coating needed to saturate a protein-adsorptive
support depends on the particle diameter of the support and is independent
of pore diameter if the protein is large enough. A well known result from
calculus is that the fractional volume in a thin outer shell is given by
6t/D, where t is the shell thickness and D is the particle diameter absent
shell. The t/D dependence also holds for non-spherical particles, in which
case average values for t and D apply. The amount of protein in a
saturated coating may be determined by experiment. In the preferred method
of the instant invention, application of dissolved BSA equal to 5 weight
percent of the silica provides a sufficient excess of BSA to saturatively
coat silica of 4.5 micrometer particle diameter and 60 angstrom pore size,
although somewhat lower amounts of BSA also work. Preferred are saturated
coatings deposited from aqueous solutions buffered near the IEP. Coatings
deposited far from the IEP adsorb additional BSA when the pH nears the
IEP. In contrast, coatings formed near the IEP remain resistant to further
protein adsorption both at and away from the IEP.
Preferred is preparation of the coating where the support is confined in a
tube so that support may be wetted by solvent or solvents, receive protein
deposition, be washed to remove protein in solution, and be brought into
contact with crosslinking agents by simply displacing each liquid that
coresides with the support in the tube by the next liquid. Such liquids
may be passed slowly through the tube once and discarded, or they may be
recycled through the tube to provide the proper exposure time with more
efficient use of the liquid and its protein or reagents.
Most preferred is preparation of the coating where these procedural steps
are performed in a stirred slurry of support particles in liquid. Exposure
of support to the next step can take place by resuspension of support
following solids recovery by filtration, sedimentation, or centrifugation,
or sometimes by simply adding a component to the slurry.
Thus, it is the object of the first treatment step of the present invention
to produce a thin but saturated physisorbed coating of protein on the
external surfaces of the porous silica support. The above-mentioned
proteins will form a saturated coating at least under vigorously contacted
conditions for exposure times typical of those encountered in use. In any
event, after the saturated coating has been applied, it is then washed.
Once that has been accomplished, then, as a third step the protein is
crosslinked.
Any conventional crosslinking reaction/agent may be used, including those
which use heat, or oxidizing agents, or chemical crosslinking agents, but
the latter are preferred. For examples, of the latter, see S. S. Wong,
Chemistry of Protein Conjugation and Cross-Linking, CRC Press, N.Y.
(1991). Often crosslinking procedures involve themselves several steps. At
least the first portion of the crosslinking reaction should be conducted
in a media and at reactant concentrations that do not displace the
adsorbed protein into solution prior to the coating being rendered
permanently insoluble by sufficient crosslinking. It is well known that
the adsorption of water soluble proteins to packing materials with some
reverse phase character is maximized at both minimum and maximum organic
cosolvent content in the mobile phase. See e.g., K. P. Hupe, "Fundamental
Chromatographic Relationships", High Performance Liquid Chromatography in
Biochemistry, ed. A. Henschen et al, Publ. VCH (Deerfield Beach, Fla.
1985) pp 37-39. Such proteins are generally also very insoluble in
solvents not miscible with water. Thus preferred reaction media ar | | |
