U.S. patent application number 09/226941 was filed with the patent office on 2001-12-13 for method for screening chromatographic adsorbents.
Invention is credited to GANAPATI, BHAT, PROTOPOPOVA, MARINA, WELCH, CHRISTOPHER J..
Application Number | 20010050254 09/226941 |
Document ID | / |
Family ID | 26751596 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010050254 |
Kind Code |
A1 |
WELCH, CHRISTOPHER J. ; et
al. |
December 13, 2001 |
METHOD FOR SCREENING CHROMATOGRAPHIC ADSORBENTS
Abstract
The invention discloses a method for rapid identification of a
candidate selective separation material by placing small samples of
the candidate material in an array of vials and adding a solution
of the analytes to be separated. The solution is allowed to
interact or equilibrate and the distribution of the analytes in the
solid or liquid phase is measured usually by gas or liquid
chromatography. The identified candidate material with the greatest
differential adsorption of the analytes is selected and used as an
adsorbent for large scale separation. The rapid screening of
chromatographic adsorbents provides an efficient way of finding
suitable absorbent materials for large scale separations.
Inventors: |
WELCH, CHRISTOPHER J.;
(GLENVIEW, IL) ; PROTOPOPOVA, MARINA; (CHICAGO,
IL) ; GANAPATI, BHAT; (GRAYSLAKE, IL) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Family ID: |
26751596 |
Appl. No.: |
09/226941 |
Filed: |
January 8, 1999 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60070887 |
Jan 9, 1998 |
|
|
|
Current U.S.
Class: |
210/635 ;
210/198.2; 210/656; 436/161 |
Current CPC
Class: |
B01J 2219/00308
20130101; B01J 20/3253 20130101; B01J 2219/00283 20130101; C40B
60/14 20130101; B01J 20/3242 20130101; B01J 2220/54 20130101; B01J
20/3261 20130101; B01J 20/286 20130101; G01N 30/89 20130101; B01D
15/08 20130101; G01N 30/48 20130101 |
Class at
Publication: |
210/635 ;
436/161; 210/198.2; 210/656 |
International
Class: |
B01D 015/08 |
Claims
What is claimed is:
1. A method for identifying a selective adsorbent for separating
analyte mixtures comprising: a. providing an array of containers
each having a candidate selective adsorbent; b. adding a solution
of the analyte mixture to be separated to each selective adsorbent
candidate in the containers; c. allowing the analyte mixture to
interact with the selective adsorbent candidate; and d. identifying
the distribution of analyte mixture in the solution or on the
selective adsorbent candidate.
2. The method of claim 1 wherein the selective adsorbent is silica
having an organic molecule covalently linked thereto.
3. The method of claim 1 wherein the selective adsorbent is
polystyrene having an organic molecule covalently linked
thereto
4. The method of claim 2 wherein the selective adsorbent is derived
from silica having an aminoalkyl group covalently linked
thereto.
5. The method of claim 4 wherein the aminoalkyl group has at least
one enantioenriched amino acid covalently linked thereto.
6. The method of claim 1 wherein the amount of selective adsorbent
is about 1 mg to 100 mg.
7. The method of claim 1 wherein the analyte mixture is tested in
an array of 50-1000 candidate selective adsorbents.
8. The method of claim 1 wherein the distribution of analytes is
measured by high pressure liquid chromatography or gas
chromatography.
9. The method of claim 8 wherein chromatographic detection by mass
spectrometry is employed.
10. The method of claim 8 wherein an aliquot of the analyte mixture
is removed and subjected to chemical derivatization prior to
analysis.
11. The method according to claim 1 wherein the analyte mixture is
a mixture of enantiomers.
12. The method of claim 1 wherein the distribution of analytes is
measured by chiroptical spectroscopy techniques such as circular
dichroism spectroscopy or measurement of optical rotation.
Description
[0001] This application claims the priority of Provisional
Application 60/070,887 filed Jan. 9, 1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to the field of separation of
molecules using selective adsorbents.
[0004] 2. Description of the Art
[0005] Since its discovery by Tswett nearly a century ago, the
technique of adsorption chromatography has evolved into a tool of
fundamental importance to the biological and chemical sciences.
Early chromatographers employed readily available adsorbents such
as calcium carbonate, sugar, starch, paper, wool, silk, alumina and
silica to perform an impressive variety of separations. Today,
researchers with a problem separation are faced with a variety of
adsorbents from which to choose. Furthermore, additional adsorbents
can readily be prepared using combinatorial chemistry approaches.
As a general rule, the more selective adsorbents allow for more
economical chromatographic separations, with simple and inexpensive
batch adsorption separations becoming possible with extremely
selective adsorbents. A means of rapidly finding the most selective
adsorbent for a given separation task is needed.
[0006] One area where the development of highly selective
adsorbents is of great importance is the large scale separation of
enantiomers using chiral stationary phases (CSPs). The current
selection of commercial chiral stationary phases (CSPs) for large
scale chromatographic separations is rather limited, and most have
been developed as general purpose CSPs rather than the best CSP for
a particular separation. While new CSPs can be designed, the
development time is often too long to merit serious consideration
by process engineers.
[0007] Within the past decade the technique of chromatographic
enantioseparation has become the method of choice for analytical
determinations of enantiopurity. Allenmark, Chromatographic
Enantioseparation: Methods and Applications, Ellis Horwood, New
York, 1991. The method is widely used, particularly in the
pharmaceutical industry, where most new chiral drugs are
manufactured in enantiomerically pure form. In recent years the use
of preparative chromatographic enantioseparation has become
increasingly popular. While generally more expensive than
manufacturing routes employing enantioselective synthesis or
classical resolution, chiral HPLC offers a considerable advantage
of speed. Consequently, many pharmaceutical companies use
preparative chiral HPLC in the early stages of drug discovery to
rapidly produce enantiomerically pure drug candidates for animal
testing, metabolism and toxicology studies, etc. Once a drug
candidate has been selected for larger scale development,
alternative manufacturing methods are often used, although in a few
cases chiral HPLC is used to produce enantiopure drugs on large
scale.
[0008] Most commercial CSPs have been developed using trial and
error methodology, and have been commercialized because they
demonstrate some general ability to separate enantiomers. Of these
many commercial CSPs, only a small fraction are available in bulk
or can be produced in an economical fashion for large scale
preparative chromatography. Francotte, E., J Chromatogr., 666,
565-601, 1994. Furthermore, rather than a CSP which has a general
ability to separate the enantiomers of a large number of racemates,
the process engineer considering a potential manufacturing route
for an enantiopure drug is interested in a CSP which can separate
the enantiomers of one particular compound.
[0009] Practical large scale chromatographic enantioseparation
requires highly enantioselective CSPs. For example, chromatographic
resolution of the enantiomers of a racemate using a CSP with an
enantioselectivity of 1.3 can be rather tedious. A comparable CSP
having an enantioselectivity of 2 can sometimes afford 5-10 fold
greater productivity.
SUMMARY OF THE INVENTION
[0010] The present invention relates to a process for screening
candidate selective adsorbents for differential adsorption of two
or more chemical components. In this process a solid phase
consisting of the candidate adsorbent is allowed to contact a
solution phase containing the component or components of interest.
Interaction or equilibration of material in the solution phase with
the stationary phase of the selective adsorbent results in a change
of concentration of the analyte or analytes in both the stationary
phase and solution phase. This change in concentration can be
measured by a variety of techniques and gives an indication of the
degree of adsorption of the analyte by the stationary phase. Thus,
small amounts of candidate selective adsorbents are placed in an
array of containers and a solution of the chemical compounds to be
separated is added to each container. The components are allowed to
interact or equilibrate with the selective adsorbent and the amount
of each component in the solution phase or in the solid phase of
the array of containers is measured. The adsorbent showing the
greatest differential adsorption for the chemical components is
identified as being potentially useful for large scale separations.
The invention is particularly useful in identifying selective
adsorbents for enantiomer separations.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 is an expanded view of the solid phase prior to
equilibration.
[0012] FIG. 2 is an expanded view of the liquid phase prior to
equilibration.
[0013] FIG. 3 is a view of the liquid and solid phase after
equilibration.
[0014] FIG. 4 shows that the performance of the DNB Leu CSP
prepared by solid phase synthesis is comparable with the
performance of the commercial version of this CSP.
[0015] FIG. 5 shows a variety of DNB-peptido CSPs prepared by solid
phase synthesis.
[0016] FIG. 6 illustrates substantial differences in the
performance of structurally similar dipeptide and tripeptide
CSPs.
[0017] FIG. 7 illustrates five amino acids used to prepare a
library of 50 dipeptide DNB CSPs and the test racemate, 1, used for
evaluation.
[0018] FIG. 8 shows three representative screening chromatograms
including the blank (no CSP), a CSP which strongly adsorbs the (R)
enantiomer of the test racemate, 1, and a CSP which strongly
adsorbs the (S) enantiomer of the test racemate, 1.
[0019] FIG. 9 shows the results of a screening of a library of 50
dipeptide DNB CSPs for the separation of the enantiomers of test
racemate, 1.
[0020] FIG. 10 shows results of a screening of a focused library of
dipeptide DNB CSPs containing hydrogen-bonding sidechains in the aa
1 position and sterically bulky sidechains in the aa 2 position.
Many of these second generation CSPs are superior to the best CSPs
in the library shown in FIG. 9.
[0021] FIG. 11 shows a separation of the enantiomers of test
racemate, 1, using a conventional 4.6.times.250 mm analytical HPLC
column containing one of the best dipeptide DNB CSPs from FIG.
10.
[0022] FIG. 12 shows preparative HPLC separation of the enantiomers
of the test racemate, 1, using the column from FIG. 11.
[0023] FIG. 13 illustrates four libraries of DNB tripeptide
CSPs.
[0024] FIG. 14 illustrates the results of the screening of leucine
library from FIG. 13 for enantioselective naproxen recognition.
[0025] FIG. 15 illustrates chromatographic separation of the
enantiomers of the drug, naproxen, using the best CSP indicated by
the CSP library screening shown in FIG. 14.
[0026] FIG. 16 illustrates libraries of acyl amino acid CSPs
comprised of four different amino acids each acylated with 40
different carboxylic acids.
[0027] FIG. 17 illustrates the results of the screening of two of
the acyl amino acid libraries from FIG. 16 for enantioselective
recognition of test racemate, 1. In both instances,
3,5-dinitrobenzamide and 4-methyl, 3,5-dinitrobenzamide are shown
to be superior to other acyl groups.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] The invention encompasses a method whereby candidate
selective adsorbents can be rapidly evaluated for their potential
for carrying out the separation of a mixture of two or more
chemical components. Using this method, libraries containing small
amounts of about 1 mg to 100 mg of many different candidate
adsorbents can be rapidly evaluated using automated equipment. This
approach dramatically decreases the time required to find a
suitable selective adsorbent for a given separation. The method is
useful for finding adsorbents which can be used for the analytical
or preparative chromatographic separation of enantiomers, the
separation of impurities from pharmaceuticals or other products,
the separation of fermentation products from their associated
impurities or any process in which two or more compounds are
separated by a chromatography or any process which relies upon
differential adsorption of two or more chemical species. The method
has the added advantage that the compound mixture for which a
separation is desired can be used directly without the need for
separations, purifications, radiolabeling or other chemical
derivatization.
[0029] The screening process is depicted schematically in the
figures which follow. A small amount of a candidate absorbent is
placed in a vial or similar receptacle (FIG. 1). The expanded view
of the candidate absorbent shows two particles containing four
pendant selectors each. Any number of particles can be used, and in
contrast to chromatography, the performance of the assay does not
require the use of very small and regular particles. Indeed, there
are some advantages to be found in the use of large particles or
even a single bead. For example, since larger particles tend to
settle more rapidly and completely, the use of large particles
allows the supernatant solution to be sampled without risk of
particles clogging the syringe.
[0030] In the case where a solid phase material which
preferentially binds one enantiomer is desired (e.g. a
chromatographic chiral stationary phase) the preferred method
involves adding a solution of the racemic mixture to the candidate
chromatographic adsorbents and measuring the enantioenrichment of
either the solution phase or the stationary phase using
chromatographic techniques such as chiral HPLC, HPLC/MS, GC, CE or
spectroscopic techniques such as NMR with chiral solvating agents
or NMR analysis of diastereomeric derivatives or chiroptical
spectroscopic techniques such as CD or polarimetry. An alternative
method of performing the assay could involve analysis of a
nonracemic solution of the target analyte or could involve
independently measuring the degree of complexation of each
enantiomer.
[0031] A dilute solution containing known relative concentrations
of the mixture of the analytes of interest is then added (FIG. 2).
In this example, two analytes are represented as circles and
crosses. It is important that the analyte solution be of low enough
concentration to prevent saturation of the adsorption sites on the
chromatographic adsorbent. In addition, the polarity of the
solution phase should be such that the target molecules are neither
completely adsorbed nor completely free in solution. Equilibration
or interaction of the material in the liquid phase with the
chromatographic adsorbent may result in the preferential binding of
one of the analytes in the mixture to the chromatographic
adsorbent, resulting in a depletion of that analyte in the solution
phase.
[0032] Analysis of the relative abundance of the analytes in either
the solid phase or the solution phase gives some indication of the
degree of selectivity of the adsorbent-analyte interaction. In the
case illustrated here, a strong preference for adsorption of the
circular analyte is depicted. Those adsorbents which show the
highest degrees of selectivity are likely candidates for a
chromatographic stationary phase which may be capable of separating
the mixture of chemical components in question, FIG. 3.
[0033] This technique has several advantages over previous methods
of evaluating candidate selective adsorbents. Only a small amount,
about 1 mg to 100 mg, of the candidate adsorbent is used in an
assay, and this material need not be packed into a column or
capillary for evaluation. Furthermore, the candidate adsorbent can
be washed free of all chemical components and reused. The target
analytes can be used directly without any need for purifications,
resolutions, or synthetic operations. A variety of analytical
techniques can be used to measure the relative abundance of the
analyte molecules in either the solid phase or the solution phase.
The process is not limited to mixtures of two analytes, but could
conceivably be used to screen for e.g., an adsorbent which would
show preferential adsorption of a single desired product from a
complex mixture containing a number of different associated
impurities. Similarly, the technique could conceivably be used to
search for an adsorbent which would preferentially adsorb the
various impurities from this same complex mixture while only weakly
adsorbing the desired product. The screening process is rapid, and
is amenable to automation, which allows for high throughput
screening of libraries of new candidate chromatographic adsorbents
prepared using solid phase diversity-generating synthetic
approaches.
[0034] A variety of analytical tools can be used to determine the
relative concentrations of the analytes in the solid phase. For
example, analysis of the relative concentrations of the analytes in
the liquid phase can be performed using chromatographic techniques
such as HPLC, HPLC/MS, SFC, CE or GC or spectroscopic techniques
such as NMR or chiroptical techniques such as CD or any analytical
technique or chemical process capable of showing the absolute or
relative concentrations of the analytes in question.
[0035] Determination of the relative concentrations of the analytes
in the solid phase can be done by a variety of methods. The extent
of enrichment in the solid phase is typically greater than that in
the supernatant solution. However, these measurements are often
more difficult, usually requiring a filtration or other phase
separation before the determination of the relative concentration
of materials adsorbed onto the solid phase can be determined. A
convenient method for determining the relative concentration of the
analytes in the solid phase simply involves removal of the
supernatant layer by rapid suction filtration, followed by the
addition of a solvent which liberates most of the adsorbed material
from the solid phase, followed by analysis of the resulting
supernatant solution by HPLC or other analytical techniques
mentioned above.
[0036] Those skilled in this art will recognize that a wide variety
of solid polymeric or inorganic particles may be functionalized to
form candidate selective adsorbents using techniques and procedures
which are known from the fields of solid phase synthesis and
combinatorial chemistry. Such particles bearing pendant groups such
as amine, carboxylic acid, hydroxyl, halide, aldehyde, or thiol may
be used for attachment of one or more molecular fragments to
provide a large number of candidate selective adsorbents. Further,
by linking enantiopure moieties to functionalized solid particles,
a large number of candidate CSPs and CSP libraries can be
prepared.
[0037] Suitable candidate adsorbents are made by techniques
described in the following examples or can be purchased from Regis
Technologies, Inc., 8210 Austin Avenue, Morton Grove, Ill.
60053-0519.
EXAMPLE 1
Silica-Based Solid Phase Synthesis
[0038] Modified solid phase peptide synthesis on aminopropyl silica
particles was chosen as a preferred method for preparing
combinatorial libraries of CSPs.
Silica-Based Solid Phase Synthesis of DNB-Leu CSP
[0039] As a model study, the well known 3,5-dinitobenzoyl Leucine
(DNB-Leu) CSP was prepared on 5 g scale using the solid phase
synthesis protocol outlined in FIG. 4. The CSP thus obtained was
packed in a column which separated a group of test analytes nearly
as well as the commercial column.
Silica-Based Solid Phase Synthesis of DNB-Peptido CSPs
[0040] Preparing and evaluating a group of peptido CSPs using a
split synthesis was conducted in a manner analagous to that shown
in FIG. 4. A representative sampling of some of the CSPs which were
made and evaluated is shown in FIG. 5. Each CSPs was prepared on 5
g scale, packed into a column and evaluated chromatographically.
Two additional CSPs from this initial group are shown in FIG. 6.
These CSPs are nearly identical, differing only in one leucine
residue. Nevertheless, substantial differences in
enantioselectivity are noted for the group of test analytes.
Microscale Silica-Based Solid Phase Synthesis of CSPs
[0041] The foregoing experiments show the utility of a silica based
solid phase synthesis approach to CSP development. While the cost
and time required to make each of these materials on 5 g scale is
less than that of conventional CSP development, an even more rapid
way of sampling the structural diversity of the DNB peptide family
was required. Consequently, candidate CSPs on 50 mg scale were
prepared and screened ex-column to evaluate the enantioselectivity
of each CSP.
[0042] A library of 50 dipeptide DNB CSPs were prepared using
combinations of the 5 amino acids; valine, glutamine,
phenylalanine, phenylglycine and proline (FIG. 7). This set
includes sterically bulky, strong hydrogen bonding and aromatic
amino acids.
[0043] The solid phase peptide synthesis which was used in the
multigram scale preparation of the CSPs shown in FIGS. 5 and 6 was
scaled down to prepare 50 mg of each of 50 dipeptide DNB CSPs
resulting from combinations of the 5 amino acids shown in FIG.
7.
Evaluation of CSP Library
[0044] The CSP library was first evaluated using the test racemate,
1. The evaluation procedure consists of adding 1 ml of a
1.times.10.sup.-5 M solution of the test racemate in 20% IPA/hexane
to each of the 50 CSP-containing vials. The vials were then capped
and transferred to an HPLC autosampler, where they were allowed to
sit for a period of 30 min. HPLC analysis of 50 .mu.l of the
supernatant solution from each vial was performed using a
46.times.250 mm (S) DNB-Leucine CSP operating at a flow rate of 1
ml/min with a mobile phase of methanol and detection at 254 nm.
Three representative chromatograms are shown in FIG. 8, including
the blank (no CSP), a CSP which strongly adsorbs the (R) enantiomer
of the test racemate, and a CSP which strongly adsorbs the (S)
enantiomer of the test racemate. The results of the screen are
presented in FIG. 9. The vertical axis in FIG. 9 represents
enantioselectivity, with the tallest bars indicating the most
enantioselective CSPs. The overall method provides useful
information on the separation capability of each material. Previous
experience with this chiral recognition system had led us to
believe that an amide hydrogen on the amino acid closest to the DNB
group (aa 2) is essential for good separation. Furthermore, it was
suspected that amino acids with a large steric group at this
position should work best, with aromatic groups at this position
generally being poorer than steric groups. It thus comes as no
surprise that the proline in position aa 2 works very poorly, while
valine and phenylalanine in this position work best. Some
unexpected results are obtained, even though this chiral
recognition system has been extensively studied for more than a
decade by a variety of techniques in addition to chromatography,
including X-ray analysis of co-crystals and nOe NMR analyses of 1:1
complexes. One unexpected result of the screen is the finding that
glutamine in position aa 1 seems to have a beneficial effect on
enantioselectivity.
Preparation and Evaluation of a Focused CSP Library
[0045] This initial screen provides a basis for further
optimization for this chiral recognition system. The initial screen
indicates that DNB dipeptide CSPs having a strong hydrogen bonding
sidechain in the aa 1 position and a sterically bulky sidechain in
the aa 2 position work best for the test analyte. A focused library
based on this motif was prepared and evaluated. As shown in FIG.
10, many of the members of this new library show superior
enantioselectivity to the DNB Val-Gln CSP, which was the best CSP
in the initial library.
Selection Scale-Up, and Evaluation of an `Optimal` CSP
[0046] One of the preferred CSPs shown in FIG. 10 was prepared on 5
g scale and packed into 4.6.times.250 mm HPLC column for
evaluation. As shown in FIG. 11, this HPLC column was shown to
separate the enantiomers of the test analyte, 1, with an
enantioselectivity in excess of 20. This HPLC column was shown to
be highly effective for the preparative separation of the
enantiomers of the test analyte, 1, as shown in FIG. 12. In this
example, near baseline resolution of enantiomers is observed, even
with a single injection of 100 mg of racemate. Analysis of the two
fractions from this preparative separation shows that the collected
enantiomers are isolated in a highly enantioenriched form.
Furthermore, the relatively rapid separation time permits a very
high preparative throughput.
[0047] This example illustrates the utility of the technology for
the discovery of a highly selective adsorbent for a given
separation problem.
EXAMPLE 2
[0048] Using an approach analogous to that described in Example 1,
a series of tripeptide DNB CSPs were prepared and evaluated. Four
such libraries of 36 CSPs each were prepared by analogous solid
phase synthesis techniques and are shown in FIG. 13. Evaluation of
this CSP library as candidate adsorbents for separation of the
enantiomers of the drug, naproxen, revealed several promising
library members, as shown in FIG. 14. FIG. 15 shows the evaluation
of the best CSP indicated by the library screening shown in FIG. 14
using a 4.6.times.250 mm HPLC column.
EXAMPLE 3
[0049] Using an approach analogous to that described in Example 1,
the series of acyl amino acid CSPs shown in FIG. 16 were prepared.
Several different BOC amino acids were coupled with
aminopropylsilica, followed by deprotection to afford the
corresponding CSPs bearing a free terminal amino group. These CSPs
were next transferred to individual vials, where they were coupled
with each of a group of 40 different carboxylic acids. The
resulting library of acyl amino acid derived CSPs was screened for
ability to separate the enantiomers of the test racemate, 1. The
results of the screens for two such sub-libraries are shown in FIG.
17. These results emphasize the fact that 3,5 dinitrobenzamide
groups works well for separation of the enantiomers of test
racemate, 1.
EXAMPLE 4
[0050] This example illustrates that the technique is not limited
to CSP libraries on a silica surface. We have prepared and
evaluated a subset of the library illustrated in FIG. 9 using
polystyrene based media. In this example, Chiron SynPhase.TM.
Crowns (PS Crown Type:I series: aminomethylated) were used to
prepare several CSPs in the dipeptide DNB series. Evaluation of the
resulting Crown CSPs showed results which were similar to those
found in Example 1, although some differences were noted. The use
of polystyrene as a solid phase may be of some use for the
preparation of adsorbent libraries owing to the fact that many
types of solid phase synthesis are possible on polystyrene or other
media which are not possible with silica. Furthermore, existing
solid phase libraries can be accessed and evaluated as candidate
adsorbents.
EXAMPLE 5
[0051] Several members of the CSP library described in Example 1
were evaluated for their ability to selectively adsorb the
enantiomers of the test racemate, 1, using HPLC with MS detection.
The evaluation procedure was the same as that described in Example
1, except that HPLC evaluation was performed using a 46.times.250
mm (R) DNB-Phenylglycine CSP operating at a flow rate of 1 ml/min
with a mobile phase of 1:1:1 methanol/acetonitrile/water with
detection by mass spectrometry. This detection method was shown to
afford essentially the same information obtained using UV
detection, and in other cases where the analyte under investigation
has poor UV absorbance, HPLC with MS detection has proven to afford
the requisite sensitivity and reliability for direct screening of
the CSP libraries.
EXAMPLE 6
[0052] An indirect chemical derivatization method was used to
evaluate several CSP libraries for their ability to separate the
enantiomers of a racemic secondary amine which had poor UV
absorbance and was not well separated by chiral HPLC. A 10.sup.-4 M
solution of the racemic secondary amine in 5% IPA/hexane was added
to a group of vials, each containing about 50 mg of a different
candidate CSPs on a porous silica support. After waiting for one
hour, 500 .mu.l of supernatant solution was withdrawn from each
vial and transferred to a fresh autosampler vial.
3,5-dinitrobenzoyl chloride (5.5.times.10.sup.-7 moles) and
diisopropylethylamine chloride (6.times.10.sup.-7 moles) were then
added to each vial. After two hours of reaction, the contents of
each vial was analyzed using an autosampler HPLC system with UV
detection.
[0053] These examples illustrate the invention and are not intended
to limit in spirit or scope.
* * * * *