Note: Descriptions are shown in the official language in which they were submitted.
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NEW CHIRAL COLUMNS WITH BROAD CHIRAL SELECTIVITY
Government Support
This invention was made in connection witll Grant Numbers NIH 1 RO 1 GM63 812-
01 and
NIH 1 R01 GM60637-01A1, from the National Institutes of Health. The United
States
Government has rights to this invention.
Field of the Invention
The present invention relates to the field of chiral chemistry. More
particularly, the present
invention relates to the separation of enantiomers, i.e., those isomers in
which the arrangement of
atoms or groups is such that the two molecules are not superimposable.
The present inventor has developed a new class of chiral columns that can
resolve a large
number of racemic compounds. These columns are stable and can be used with a
number of
mobile phase solvents.
Background of the Invention
Stereoisomers are those molecules which differ from each other only in the way
their
atoms are oriented in space. Stereoisomers are generally classified as
diastereomers or
enantiomers; the latter embracing those which are mirror-iinages of each
other, the former being
those which are not. The particular arrangement of atoms that characterize a
particular
stereoisomer is known as its optical configuration, specified by known
sequencing rules as, for
example, either + or - (also D or L) and/or R or S.
Though differing only in orientation, the practical effects of stereoisomerism
are
important. For example, the biological and pharmaceutical activities of many
compounds are
strongly influenced by the particular configuration involved. Indeed, many
compounds are only of
widespread utility when employed in a given stereoisomeric form.
Living organisms usually produce only one enantiomer of a pair. Thus only (-)-
2-methyl-
1-butanol is forined in yeast ferinentation of starches; only (+)-lactic acid
is formed in the
contraction of muscle; fruit juices contain only (-)-malic acid, and only (-)-
quinine is obtained
from the cinchona tree. In biological systems, stereochemical specificity is
the rule rather than the
exception, since the catalytic enzymes, which are so important in such
systems, are optically
active. For example, the sugar (+)-glucose plays an important role in animal
metabolism and is the
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basic raw material in the fermentation industry; however, its optical
counterpart, or antipode, (-)-
glucose, is neither metabolized by animals nor fermented by yeasts. Other
examples in this regard
include the mold Penicillium glaucum, which will only consume the (+)-
enantiomer of the
enantiomeric mixture of tartaric acid, leaving the (-)-enantiomer intact.
Also, only one
stereoisomer of chloromycetin is an antibiotic; and (+)-ephedrine not only
does not have any drug
activity, but it interferes with the drug activity of its antipode. Finally,
in the world of essences,
the enantiomer (-)-carvone provides oil of spearmint with its distinctive
odor, while its optical
counterpart (+)-carvone provides the essence of caraway.
Thus, as enzymes and other biological receptor molecules possess chiral
structures,
enantioiners of a racemic compound may be absorbed, activated, and degraded by
them in
different manners. This phenomenon causes that in many instances, two
enantiomers of a racemic
drug may have different or even opposite pharmacological activities. In order
to aclcnowledge
these differing effects, the biological activity of each enantiomer often
needs to be studied
separately. This and other factors within the pharmaceutical industry have
contributed
significantly to the need for enantiomerically pure compounds and thus the
need for chiral
chromatography.
Accordingly, it is desirable and oftentimes essential to separate
stereoisomers in order to
obtain the useful version of a coinpound that is optically active.
Separation in this regard is generally not a problem when diastereomers are
involved:
diastereomers have different physical properties, such as melting points,
boiling points,
solubilities in a given solvent, densities, refractive indices etc. Hence,
diastereomers are normally
separated from one another by conventional metliods, such as fractional
distillation, fractional
crystallization or chromatography.
Enantiomers, on the other hand, present a special problem because their
physical
properties are identical. Thus they cannot as a rule--and especially so when
in the form of a
racemic mixture--be separated by ordinary methods: not by fractional
distillation, because their
boiling points are identical; not by conventional crystallization because
(unless the solvent is
optically active) their solubilities are identical; not by conventional
chromatography because
(unless the adsorbent is optically active) they are held equally onto the
adsorbent. The problem of
separating enantiomers is further exacerbated by the fact that conventional
synthetic techniques
almost always produce a mixture of enantiomers. When a mixture comprises equal
amounts of
enantiomers having opposite optical configurations, it is called a racemate;
separation of a
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racemate into its respective enantiomers is generally known as a resolution,
and is a process of
considerable importance.
Chiral columns that can resolve a large number of racemic compounds (general
chiral
columns) are in high demand. They are needed routinely in many laboratories,
especially in
pharmaceutical industry. Prior to the present invention, Daicel columns,
macrocyclic antibiotic
columns, and the Whelk-O coluinns were probably known as the industrial
leaders in this type of
general chiral columns. The present inventor has developed a new class of
general chiral columns
based on the use of proline and its analogues.
Furthermore; and importantly, the columns of the present invention have the
capability of
resolving at least a similar or higher percentage of the compounds tested.
Furthermore, the
columns of the present invention provide better separation on some of the
compounds tested and
can resolve certain compounds that cannot be resolved with the commonly used
commercial
columns listed above.
The columns of the present inventions are stable and can be used with a large
number of
mobile phase solvents. Therefore, the columns of the present invention should
find important
applications as general chiral columns.
A large number of chiral columns have been prepared in the past; however, only
a few
demonstrated broad chiral selectivity. As stated above, the successful
examples include the
popular Daicel coluinns, the Chirobiotic columns, and the Whelk-O1/2 columns.
The Daicel
colunms are prepared by coating sugar derivatives onto silica gel. Chirobiotic
columns are
prepared by immobilizing macrocyclic glycopeptides onto silica gel. Whelk-O
1/2 columns
contain both electron rich and electron deficient aromatics. These columns
have broad chiral
selectivity and have been applied successfully to resolve a fair number of
racemic compounds.
They have different selectivity and stability profiles. Their selectivities
complement each other in
some cases, while they duplicate each other in other cases. Some of the
columns are more suited
for reversed phase conditions and others for normal phase conditions. Each
column has its own
strengtlis and weaknesses. Despite these progresses, there are still many
compounds that cannot
be resolved or resolved well using these commercial available columns.
Therefore, there is still a
significant need to develop new columns that have relatively broad chiral
selectivity.
Summary of the Invention
The present invention is directed to a chiral selector that represents an
improvement in the
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art of enantiomeric separation. Tlius, one embodiment of the present invention
is a general chiral
column with a multiple proline-based chiral selector.
Another embodiment of the present invention is a chiral stationary phase made
of peptides
with 2 or more prolines, including chiral selectors with 2, 3, 4, 5, 6, or 10
prolines. Also included
within the scope of the present invention are analogs and isomers of prolines,
and analogs and
isomers of the chiral selector compounds of the present invention.
Another embodiment of the present invention is a chiral stationary phase (or
column) of
the following formula:
end-capping
Pro
group linker Support
n
wherein n is any integer of 2 or greater, and analogs and isomers thereof.
Another embodiment of
the present invention is where n is any integer from 2-10.
The separations achieved for analytes are comparable or superior to those
achieved on
Daicel AD, Daicel OD, and Whelk 02 columns. The multiple proline-based chiral
columns of the
present invention show promise as a superior general chiral column.
Brief Description of the Drawings
Figure 1 shows the structure for amino acid L-Proline and its associated
stationary phases
Fmoc-Pro-(Me)Ahx-APS (CSP1), Fmoc-Pro2-(Me)Ahx-APS (CSP2), Fmoc-Pro4-(Me)Ahx-
APS
(CSP3); and Fmoc-Pro6-(Me)Ahx-APS (CSP4). CSP2-4 are embodiments of compounds
of the
present invention.
Figure 2 shows the synthesis of one embodiment of the present invention, Fmoc-
Pro4-
(Me)Ahx-APS chiral stationary phase (CSP3): Synthesis of Fmoc-Pro4-(Me)Ahx-APS
chiral
stationary phase (CSP3): (a) Fmoc-(Me)Ahx-OH, DIC; (b) (1) Piperidine; (2)
Fmoc-Pro-OH,
HATU; (c) AcOH; (d) aminopropyl-silica gel, HATU.
Description of the Invention
The present inventor has developed a new chiral column that has relatively
broad chiral
selectivity, when compared witli Daicel columns and Whelk 02 column, as
industry standards or
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industry models. Additionally, the chiral columns of the present invention are
stable in a number
of mobile phase conditions.
The success rate of the chiral column of the present invention compares well
with the best
commercially available general chiral columns developed over the last few
decades. For 22
racemic compounds chosen based on their availability (see example 4), our Pro4
column (CSP 3)
resolved 17 compounds; our Pro2 column (CSP2) resolved 16 compounds; our Pro6
column
(CSP4) resolved 15 compounds. In comparison, Daicel OD column resolved 18,
Daicel AD
resolved 16, and Whelk-02 resolved 15 compounds. The monoproline column (CSP1)
is much
less effective, as it can resolve only 6 out of the 22 compounds tested. The
achieved resolutions
with the monoproline column are also very modest.
Proline is a unique amino acid in many ways (Figure 1). Instead of having a
primary
amino group as in other a-amino acids, it contains a secondary amine. Because
of the cyclic
structure, rotation around the nitrogen-a-carbon bond is restricted. Also
because of the cyclic
structure, proline is not ideally suited for a-helix or P-sheet conformation;
instead, polyproline
forms its own unique helical conformation (Polyproline I and polyproline II).
The ainide bond in
polyproline is sterically hindered compared with other oligopeptides. The
distinctly different
conformational and structural features of polyprolines suggest that they may
behave quite
differently from other short oligopeptides that have been studied in chiral
chromatography.
The present inventors discovered that proline based chiral selectors,
including the
embodiment tetraproline based chiral stationary phase 3 (Figure 1) , diproline
based chiral
stationary phase 2, hexaproline based chiral stationary phase 4 have
relatively broad chiral
selectivity, while mono-proline stationary phase 1 is largely ineffective.
Immobilization of the chiral selectors'of the present invention to silica gel
is accomplished
through a linker group. One example of a linker group of the present invention
is a N-alkylamino
group. A second example is a N-methylamino group. Another example is 6-N-
methylaminohexanoic acid. The amide bond between these linlcers and proline
residue is more
sterically hindered due to the N-methyl or N-alkyl group. The particular
linker group can be
selected by one of ordinary skill in the art depending on the analyte to be
tested. For example,
when the selector Fmoc-Pro-Pro is immobilized using 6-N-methylaminohexanoic
acid, it may
resolve about 16 out of about 22 analytes tested. For the same chiral
selector, when immobilized
using 6-aminohexanoic acid, it resolved only 4 out of the same group of
analytes.
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Additionally, the stationary phase compounds of the present invention may
comprise
various end-capping groups as known in the art.
By use of the term proline with respect to the present invention, it is
understood that
analogs and isomers of proline are included. For example all stereoisomers are
included.
Additionally, analogs are included. Examples of the analogs that are included
herein are those
with the following skeleton structure feature such as in D-proline,
hydroxyproline, and pipecolinic
acid:
X
n
H Chiral Center
wherein n is an integer (such as 1, 2, 3, 4, 5, etc.) and X is a heteroatom
such as O,S,N; and other
unspecified atoms can be carbon or heteroatoms.
These covalently bound columns of the present invention are stable in common
organic
solvents, including CH2C12 and CHC13. Therefore, a wide selection of mobile
phase conditions
could be applied in method development. For several analytes, the present
inventor attempted
resolution with CHzCl2/hexane as the mobile phase and effective separation was
also achieved
(example 6). Wider solvent choices have advantages in that some racemic
analytes are soluble in
only certain solvents and some compounds can be resolved better in certain
solvents
In terms of potential interaction modes with the analytes, examples of the
chiral selectors of
the present invention are forming attractive hydrogen bonds with the analyte
and they may also
have attractive polar interactions witli the analyte. In addition, steric
interaction between analyte
and chiral selector could also be important.
The following examples and experimental section are designed to be purely
exemplary in
nature. Thus, this section should not be viewed as being limiting of the
present invention.
Examples
Throughout this section, various abbreviations are used, including the
following: DIC,
diisopropylcarbodiimide; HATU, O-(7-Azabenzotriazol-1-yl)N,N,N',N'-
tetramethyluronium
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hexafluorophosphate; DIPEA, N,N-Diisopropylethylamine; DMF, N,N-
Dimethylformamide;
DCM, Dichloromethane; DMAP, 4-(dimethylaminopyridine); NMM: N-
methylmorpholine;
Fmoc, 9-Fluorenylmethoxycarbonyl; (Me)Ahx: 6-methylaminohexanoic acid; Fmoc-
(Me)Ahx-
OH, 6-[(9H-fluoren-9-ylmethoxy)carbonyl]methylamino hexanoic Acid; Fmoc-Ahx-
OH, 6-[(9H-
fluoren-9-ylmethoxy)carbonyl]aminohexanoic acid; Fmoc-Pro-OH, N-a-Fmoc-L-
proline..
General Supplies and Equipment:
Amino acid derivatives were purchased from NovaBiochem (San Diego, CA). All
other
chemicals and solvents were purchased from Aldrich (Milwaukee, WI), Fluka
(Ronkonkoma,
NY), or Fisher Scientific (Pittsburgh, PA). HPLC grade Kromasil silica gel
(particle size 5 m,
pore size 100 A, and surface area 298 m2/g) was purchased from Akzo Nobel (EKA
Chemicals,
Bohus, Sweden). Selecto silica gel (32-63 gm) from Fisher Scientific was used
for flash column
chromatographic purification of target compounds. Thin-layer chromatography
was completed
using EM silica ge160 F-254 TLC plates (0.25 mm; E.Merck, Merck KGaA, 64271
Darmstadt,
Germany). Elemental analyses were conducted by Atlantic Microlab, Inc.
(Norcross, GA).
HPLC analyses were completed with a Beckman analytical gradient system (System
Gold). UV
spectra were obtained with a Shimadzu UV 201 spectrometer (cell volume 3 mL;
cell pass length
10 mm).
Exatnple 1: PrepaNation of chiral stationary phase Fmoc-PNo-(Me)Ahx-APS (CSP1)
To 0.80 g of (Me)Ahx-APS silica (the surface (Me)Ahx concentration is 0.64
mmol/g) are
added mixtures of Fmoc-Pro-OH (3 equiv., 0.52 g), HATU (3 equiv., 0.58 g), and
DIPEA (3
equiv., 0.20 g) in 8 mL of DMF. After agitating for 6 h, the resulting silica
is filtered and washed
with DMF, Methanol, and DCM to yield the desired chiral stationary phase. The
surface Pro
concentration is determined to be 0.57 mmol/g based on the Fmoc cleavage
method. The
resulting chiral stationary phase is packed into a 50 x 4.6 mm HPLC column
using a standard
slurry packing method.
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Example 2: Preparation of chiral stationary phase Fmoc-Pro2-(Me)Ahx APS (CSP2)
To 0.80 g of (Me)Ahx-APS silica (the surface (Me)Ahx concentration was 0.64
mmol/g)
were added mixtures of Fmoc-Pro-OH (3 equiv., 0.52 g), HATU (3 equiv., 0.58
g), and DIPEA (3
equiv., 0.20 g) in 8 mL of DMF. After agitating for 6 h, the resulting silica
was filtered and
washed with DMF, Methanol, and DCM. The surface Pro concentration was
determined to be
0.55 mmol/g based on the Fmoc cleavage method. The Fmoc protecting group was
then removed
by treatment of the silica with 10 mL of 20% (V/V) piperidine in DMF for 1 h.
The deprotected
silica, Pro-(Me)Ahx-APS, was collected by filtration and washed with DMF,
Methanol, and
DCM. Then another module, Fmoc-Pro-OH, was coupled to the resulting silica
following an
identical reaction sequence and yielded the desired chiral selector on the
silica gel. The surface
Fmoc concentration was determined to be 0.52 mmol/g based on the Finoc
cleavage method. The
resulting chiral stationary phase was packed into a 50 x 4.6 mm HPLC column
using the standard
slurry packing method.
Example 3: Preparation of chiral stationary phase Fmoc-PN04-(Me)Ahx-APS (CSP3)
To Rink acid resin (100-200 mesh, 3.0 g, 0.43 mmol/g) preswelled with DCM (20
mL, 30
min) was added the mixture of Fmoc-(Me)Ahx-OH (1.42 g, 3.87 mmol), DMAP (0.16
g,1.29
mmol), NMM (0.39 g, 3.87 mmol), and DIC (0.49 g, 3.87 mmol) in DCM-DMF(1:1
V/V, 10
mL). After agitating for 6 h, the resin was collected by filtration and washed
with DMF, DCM,
and Methanol (20 mL x 3). The Fmoc group was then removed by treatment with 20
mL of 20%
(V/V) piperidine in DMF for 30 min. The deprotected (Me)Ahx-O-Rink resin was
collected and
washed with DMF, DCM, and Methanol (20 mL x 3).
To (Me)Ahx-O-Rink resin was added the mixture of Fmoc-Pro-OH (1.31 g, 3.87
mmol),
HATU (1.47 g, 3.87 mmol), and DIPEA (0.50 g, 3.87 mmol) in 20 mL of anhydrous
DMF. After
agitating for 3 h, the resin was filtered and washed with DMF, DCM, and
Methanol (20 mL x 3).
The Fmoc group was then removed and the second, third and fourth modules, Fmoc-
Pro-OH,
were coupled by following exactly the same procedures as described above to
yield the desired
Fmoc-(Pro)4-(Me)Ahx-O-Rink resin.
The resin was then treated with 1% TFA in DCM (20 mL, 10 min) to release Finoc-
(Pro)4-
(Me)Ahx-OH from the resin. This cleavage reaction was repeated one more time
to ensure
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complete reaction. The crude product obtained was purified by flash column
chromatography on
silica gel (mobile phase: 5% Methanol in DCM) to yield the desired Fmoc-(Pro)4-
(Me)Ahx-OH as
a white solid (0.90 g, 92%). 'H NMR (CD2C12): 8 1.2-1.7 (m, 6H), 1.9-2.4 (m,
18H), 2.80 (s, 3H),
3.2-3.6 (m, 1011), 4.2-4.7 (m, 7H), 7.1-7.6 (m, 8H), 9.6 (br, 1H). ESI-MS: m/z
756.0 (M+H").
A mixture of Fmoc-(Pro)4-(Me)Ahx-OH (0.90 g, 1.19 mmol), HATU (0.45 g, 1.19
mmol),
and DIPEA (0.15 g, 1.19 mmol) in 8 mL of anhydrous DMF was added to 0.7 g of 3-
aininopropyl
silica gel (APS). APS was prepared from Kromasil silica gel (5 m spherical
silica, 100 A, 298
m2/g) and 3-aminopropyltriethoxysilane. The surface amino concentration is
0.66 mmol/g, based
on elemental analysis data of nitrogen (C, 3.11; H, 0.83; N, 0.93). After
agitating the mixture for
4 h, the stationary phase was collected by filtration and washed with DMF,
DCM, and Methanol
(10 mL x 3). The surface Fmoc concentration was determined to be 0.27 mmol/g
based on Fmoc
cleavage method. The resulting chiral stationary phase was packed into a 50 x
4.6 mm HPLC
column using the standard slurry packing method.
The following examples set forth various chromatographic measurements.
Therein,
retention factor (k) equals to (tr-t0)/t0 in which tr is the retention time
and t0 is the dead time.
The separation factor (a) equals k2/kl, ratio of the retention factors of the
two enantiomers.
Separation factor of 1 indicates no separation. The larger the separation
factor, the better the
separation is. Dead time t0 was measured with 1,3,5-tri-t-butylbenzene as the
void volume
marker. Flow rate at 1 inL/min., UV detection at 254 nin.
Example 4
This example compares chromatographic resolution of racemic compounds with
chiral
columns, including embodiments of the present invention (Pro2 (CSP2), Pro4
CSP3), Pro6(CSP4)).
In the following table, kl is the retention factor of the least retained
enantiomer and the separation
factor (a) is defined earlier This example also shows that a mono-proline
chiral column does not
perform sufficiently.
Furthermore, this example shows embodiments of the present invention in
comparison
with known commercial columns.
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Table 1. Chromatographic resolution of racemic compounds with chiral columns.
kl is the
retention factor of the least retained enantiomer. Mobile phases are solutions
of specified
percentage of IPA and acetic acid in hexanes.
Analyte name Analyte Structure Prol Pro2 Pro4 Pro6 Daicel Daicel Whelk-
OD AD 02
Benzoin oH a:l a: 1.07 a: 1.09 a:1.12 a: 1.61 a:1.32 a: 2.12
k1:5.78 k1:8.22 k1:6.35 k1:16.0 k1:4.68 k1:3.46 k1:1.83
I\ \ 3%IPA 3%IPA 3%IPA 5%IPA 3%IPA 15%IPA 5%IPA
O
Hydrobenzoin OH / a:l a: 1.12 a: 1.13 a:1.15 a:l a: 1.08 a: 1.33
\ k1:17.7 k1:21.15 kl:17.9 k1:25.79 k1:7.35 k1:5.15 k1:4.18
I\ 1 4%IPA 8 8%IPA 4%IPA 8%IPA 4%IPA
OH 4%IPA 4%IPA
Benzoin oxime OH / a: 1 a: 1.09 a: 1.13 a: 1.20 a: 1.13 a: 1.24 a: 1.31
k1:12.2 ki:16.08 kl:15.3 k1:41.45 k1:2.82 k1:4.55 11:1.40
8 20%IPA 6 30%IPA 10%IPA 15%IPA 10%IPA
N 20%IP 20%IP
oH A A
2,2,2-Trifluoro-l- a:l a:1.28 a: 1.56 a: 1.78 a:1.13 a: 1.47 a: 1.13
(9-anthryl) I k1:16.4 k1:23.44 kl:18.4 k1:22.58 k1:1.26 k1:1.99 k1:0.62
ethanol \ \ / 0 l0%IPA 8 25%IPA 15%IPA 10%IPA 10%IPA
10%IP 10%IP
F3C OH A A
a- \ a:1 a:1.06 a:1.10 a:1.10 a:1.16 a:1.11 a:1
(pentafluoroethyl} I k1:19.3 k1:16.08 k1:8.91 k1:8.62 k1:0.90 k1:0.79 k1:0.70
a-(trifluoromethyl)- ~ 1 3%IPA 3%IPA 5%IPA 1%IPA 3%IPA 3%IPA
Benzenemethanol 3%IPA
F3C C2F5
Warfarin \ a:l a:1.11 a:1.08 a:1.18 a:2.49 a:3.94 a:1.97
I k1:13.9 k1:10.57 k1:11.1 k1:17.50 k1:6.40 k1:5.02 k1:10.06
1 10%IPA 9 10%IPA 15%IPA 20%IPA 20%IPA
OH O 10%IP & 10%IP &
A& 1% A& 1%
1% AcOH 1% AcOH
AcOH AcOH
Sec-Phenethyl a: l a: 1.02 a: 1.02 a: 1.04 a: 1.37 a: I a: 1.03
alcohol k1:6.67 k1:11.3 k1:8.07 k1:19.42 k1:8.30 k1:4.12 k1:3.64
OH 1%IPA 1%IPA 1%IPA 1%IPA 1%IPA 2%IPA 2%IPA
in in hexane in in hexane in hexane in hexane in hexane
hexane hexane
a-Methyl-2- a: 1 a: 1 a:1.04, a: 1 a:1 a: 1.05 a: 1.02
Naphthalene k1:13.3 k1:22.36 kl:17.6 kl:18.81 k1:6.25 k1:1.98 k1:4.39
methanol / I \ OH 6 1%IPA 2 3%IPA 3%IPA 10%IPA 3%IPA
1%IPA 1%IPA
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1-Acenaphthenol OH a: a:1 a: 1 a:1 a:1.16 a: 1.08 a: 1.28
k1:7.83 k1:13.36 kt:10.2 k1:28.06 k1:5.46 k1:6.58 k1:4.96
3%IPA 3%IPA 8 3%IPA 3%IPA 3%IPA 3%IPA
3%IPA
3-Phenyl-Glycidol a: 1 a:1 a: 1 a:1 a: 1.15 a: 1 a: 1.37
o k1:5.23 k1:5.64 k1:6.77 k1:10.38 k1:16.87 k1:8.01 k1:8.74
3%IPA 3%IPA 3%IPA 5%IPA 10%IPA 8%IPA 10%IPA
1,1'-si-2-naphthol a: 1.04 a: 1.16 a: 1.29 a: 1.42 a: 1.16 a: 1.13 a: 1
kt:11.4 k1:32.80 k1:23.8 k1:9.94 k1:4.49 k1:3.58 k1:1.26
OH 8 75%IPA 3 90%IPA 8%IPA 25%IPA 5%IPA
75%IP 75%IP
/ I \ OH A A
2,2'-Diol- a:1.14 a:1.17 a:1.32 a:1.67 a:1.32 a:1 a:1
5,5',6,6',7,7',8,8'- kt:12.5 k1:1 1.10 k1:10.9 kt:19.75 k1:3.98 k1:8.53
k1:4.47
octahydro-1,1'-
binaphthalene / OH 8 10%IPA 5 25%IPA 5%IPA 10%IPA 5%IPA
OH 10%IP 10%IP
A A
1,2,3,4-Tetrahydro- oMe a: 1.05 a: 1.18 a: 1.24 a: 1.20 a:1.15 a:1.40 a: 1.16
4- k 14.8 k 18.68 k 12.6 k 23.53 k 2.13 Ic 3.44 k1:2.27
4 methox~hen 1 ~ 1 ~ ~ t i
(" y)-
6-methyl-2-thioxo- I 15%IPA 0 15%IPA 15%IPA 15%IPA 15%IPA
5-pyrimidine 0 15%IP 15%IP
carboxylic acid NH A A
ethyl ester EtO
N~S
1,2,3,4-Tetrahydro- a: 1.12 a: 1.20 a: 1.41 a: 1.32 a: 1.30 a:1.36 a: 1
4- kt:44.6 k1:27.80 k1:25.0 k1:30.13 k1:2.87 k1:4.62 k1:2.23
(4-hydroxyphenyl)- I
6 50%IPA 7 70%IPA 15%IPA 15%IPA 15%IPA
6-methyl-2-thioxo-
5-Pyrimidine 30%IP 30%IP
carboxylic acid ~ A A
ethyl ester NH
Et0
N ~S
1-[1,2,3,4- OMe a:1 a: 1.20 a: 1.21 a: 1.34 a: 1.18 a:1.70 a: 1
Tetrahydro-4- k1:27.0 k1:40.60 k1:25.7 k1:33.90 k1:2.62 k1:3.62 k1:1.83
(4-methoxyphenyl)- 3 15%IPA 2 25%IPA 15%IPA 15%IPA 15%IPA
6-methyl-2-thioxo-
5-pyrimidinyl] 15%IP 15%IP
ethanone A A
Ac NH
I N~S
Hexobarbital M. a: 1 a:1 a:1 a: 1 a:1.12 a:1.46 a: 1
~ k1:28.8 k1:22.28 kt:16.9 kt:11.26 k1:6.26 lc1:2.42 k1:1.95
e~N e 6 1%IPA 8 3%IPA 5%IPA 8%IPA 5%IPA
HN 1 %IPA 1 %IPA
M.
O
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Temazepam Ph Ci a: 1.09 a:1 a:1 a:1 a:l a:1 a:1.19
N/ I\ k1:22.0 k1:25.54 k1:20.2 k1:25.52 k1:3.39 k1:4.12 k1:3.40
3 2%IPA 4 5%IPA 25%IPA 25%IPA 25%IPA
H ~ 2%IPA 2%IPA
~_N
5-Methyl- a:1 a: 1.16 a: 1.17 a: 1.34 a: 1.08 a:1 a: 1.11
5-(2,5-dichloro ~NH k1:11.2 k1:14.6 k1:8.55 k1:11.64 k,:4.29 k1:4.80 k1:1.96
phenyl) HN 1 15%IPA 15%IP 25%IPA 8%IPA 8%IPA 10%IPA
15%IP A
hydantoin A
CI
CI
5-Methyl-5-phenyl a: 1 a: 1.10 a: 1.15 a: 1.32 a: 1.09 a:l a: 1.46
hydantoin ~NH kl:16.2 k1:25.00 k1:15.6 k1:10.7 k1:4.06 k1:3.24 k1:1.67
HN 4 8%IPA 8%IPA 20%IPA 8%IPA 8%IPA 10%IPA
O 8%IPA
Mephenytoin ~_N a:1 a:1.14 a:1.14 a:1.27 a:1.10 a:1.37 a:1
k1:5.86 k1:7.86 k1:6.93 k1:9.85 k1:5.20 k1:3.56 k1:3.35
HN 2%IPA 2%IPA 2%IPA 3%IPA 4%IPA 4%IPA 4%IPA
O
Et
sec-Butyl O i a: 1 a:1 a: 1 a: a:1 a:1.04 a: 1.05
carbanilate ~N ~ I k1:3.62 k1:7.30 k1:7.64 k1:15.34 k1:6.18 k1:3.35 k1:3.36
H 1%IPA 1%IPA 1%IPA 3%IPA 2%IPA 2%IPA 1%IPA
Methyl Mandelate OH a: 1.02 a: 1.10 a: 1.17 a: 1 a: 1.25 a:1.08 a: 1.07
k1:7.97 k1:10.8 k1:8.31 k1:17.49 k1:2.82 k1:1.85 k1:1.12
COOMe 1%IPA 1%IPA 1%IPA 3%IPA 3%IPA 10%IPA 10%IPA
Example 5: Specific embodiments, for exemplary purposes, of the stationaNy
phase compounds of
the present invention and silica supports.
This example sets forth poly-proline compounds of the present invention,
including
embodiments with different end-capping groups. The end-capping groups are
bonded to the
nitrogen atom that is further away from the support. As is noted in the
example, some end-
capping groups such as pivaloyl (PIV) (CSP-6) are more effective for some
analytes tlian others,
such as TAPA. Overall, several different end-capping groups useable with the
present invention
such as Piv, Fmoc, Boc, Cbz, Aca, Dmb, Tpa all work well. CSP-5, which has no
end-capping
group, did not perform as well with respect to some analytes.
12
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Pro-Pro-N(Me)-Ahx-APS: CSP-5
NH
\ H
'---$/O O
N N_"~Si ~ Si02
Et0
Piv-Pro-Pro-N(Me)-Ahx-APS: CSP-6
~Iyo
AO
C N
0 H
N Si02
EtO
Fmoc-Pro-Pro-N(Me)-Ahx-APS:CSP-2
O~O
N
\'' O O
H
N
~ Si02
N EtO
I
0
Boc-Pro-Pro-N(Me)-Ahx-APS: CSP-7
O
N
O
0 H
N~ Si02
N Et0
0
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Cbz-Pro-Pro-N(Me)-Ahx-APS: CSP-8
p
O~O
N
O 0
\ II H
SQ1 S102
i E10 O
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Aca-Pro-Pro-N(Me)-Ahx-APS: CSP-9
0
N
O H
0
Si02
N EtO
z I
O
Tapa-Pro-Pro-N(Me)-Ahx-APS: CSP-10
NO2
02N I
NO2
o-N
--IYO NOZ
N
\ //O
/ O H
N N/\/Si~ SiOg
I EtO
I
Dinb-Pro-Pro-N(Me)-Ahx-APS: CSP-11
/ I
~ O
N
\'' O 0 H I
N~~ si ~ SiO2
EtO I
O
Tpa-Pro-Pro-N(Me)-Ahx-APS: CSP-12
Fl- 0
\\, ~! 0 H
N\~ N~/Si~ Si02
Et0
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Table 2. Impact of end-capping groups.
Analyte name CSP-5 CSP-6 CSP-2 CSP-7 CSP-8 CSP-9 CSP-10 CSP-11 CSP-12
Benzoin a: 1 a: 1.12 a: 1.07 a: 1.07 a: 1.08 a: 1 a: 1 a: 1.10 a: 1.07
k1:5.84 k1:6.34 k1:8.22 k1:4.45 k1:4.63 k1:7.71 k1:19.0 k1:7.76 k1:6.00
3%IPA 3%IPA 3%IPA 3%IPA 3%IPA 3%IPA 2 3%IPA 3%IPA
3%IPA
Hydrobenzoin a: 1.07 a: 1.22 a: 1.12 a: 1.11 a: 1.11 a: 1.10 a:1.14 a:1.13
a:1.16
kl:16.7 k1:17.0 k1:21.15 k1:10.71 k1:13.33 k1:16.8 k1:26.4 kl:18.09 k1:15.17
1 0 4%IPA 4%IPA 4%IPA 1 1 4%IPA 4%IPA
4%IPA 4%IPA 4%IPA 4%IPA
Benzoin oxime a: 1 a: 1.12 a: 1.09 a: 1 a: 1 a: 1 a: 1 a: 1.10 a: 1.08
k1:11.4 k1:14.6 k1:16.08 k1:10.09 k1:12.23 kl:13.4 k1:16.0 1c1:15.00 k1:11.71
4 5 20%IPA 20%IPA 20%IPA 4 3 20%IPA 20%IPA
20%IP 20%IP 20%IP 20%IP
A A A A
2,2,2-Trifluoro-l- a: 1 a: 1.58 a: 1.28 a: 1.28 a: 1.33 a:1.16 a: 1.28 a: 1.30
a:1.40
(9-anthryl) k1:17.2 k1:22.4 k1:23.44 k1:15.08 kl:15.82 k1:20.0 k1:31.5
k1:20.47 k1:16.03
ethanol 3 10%IP 10%IPA 10%IPA 10%IPA 2 9 10%IPA 10%IPA
10%IP A 10%IP 40%IP
A A A
a- a: 1 a:1.14 a: 1.06 a: 1.11 a: 1.10 a: 1.06 a: 1.10 a: 1.09 a: 1.09
(pentafluoroethyl) k1:9.72 k1:8.89 k1:16.08 k1:7.55 k1:5.76 k1:6.18 k1:8.13
k1:13.80 k1:5.75
3%IPA 3%IPA 3%IPA 3%IPA 3%IPA 3%IPA 3%IPA 3%IPA 3%IPA
a-
(trifluoromethyl)-
Benzenemethanol
Warfarin a: 1 a: 1.20 a: 1.11 a:1 a:1.16 a: 1.12 a:1 a: 1.16 a: 1.14
k1:41.1 k1:12.4 k1:10.57 k1:19.53 kl:11.55 k1:14.3 k1:28.6 kl:13.41 k1:12.68
0 1 10%IPA 25%IPA 10%IPA 4 1 25%IPA 10%IPA
90%IP 10%IP & & 10%IP 10%IP &
A A& 1% 1% A& A& 1%
1% AcOH AcOH 1% 1% AcOH
AcOH AcOH AcOH
Sec-Phenethyl a:1 a: 1.08 a:1.02 a: 1.03 a: 1 a: 1 a: 1 a: 1 a: 1
alcohol k1:6.47 k1:8.42 k1:11.3 k1:6.30 k1:6.02 k1:6.84 k1:9.37 k1:19.34
k1:1.90
1%IPA 1%IPA 1%IPA 1%IPA 1%IPA 1%IPA 1%IPA 1%IPA 1%IPA
a-Methyl-2- a:1 a:1 a:1 a:1 a:1.02 a:1.10 a:1 a:1.04 a:1
Naphthalene k1:15.3 k1:17.3 k1:22.36 k1:13.07 kl:12.96 k1:14.8 k1:17.1 k1:7.65
k1:1.19
methanol 0 1 1%IPA 1%IPA 1%IPA 9 5 1%IPA 1%IPA
1 %IPA 1 %IPA 1 %IPA 5%IPA
1-Acenaphthenol a: 1 a: 1 a: 1 a:1 a: 1 a: 1 a: 1 a:1 a:1
k1:8.12 k1:10.7 k1:13.36 k1:7.34 k1:8.45 kj:11.0 k1:21.4 k1:13.90 k1:8.95
3%IPA 8 3%IPA 3%IPA 3%IPA 4 1 3%IPA 3%IPA
3%IPA 3%IPA 7%IPA
3-Phenyl- a: 1 a:1 a: 1 a: 1 a: 1 a: 1 a: 1 a:1 a:1
Glycidol k1:9.54 k1:11.1 k1:5.64 k1:10.75 k1:9.12 k1:11.2 k1:20.8 k1:15.49
k1:9.78
3%IPA 3 3%IPA 3%IPA 3%IPA 9 5 3%IPA 3%IPA
3%IPA 3%IPA 3%IPA
1,1'-Bi-2- a: 1.05 a: 1.34 a: 1.16 a: 1.17 a: 1.17 a: 1.35 a: 1 a: 1.18 a:
1.30
naphthol k1:13.3 k1:20.9 k1:32.80 kl:13.21 k1:17.07 ki:15.7 k1:28.6 k1:21.89
k1:19.51
1 0 75%IPA 75%IPA 75%IPA 3 3 75%IPA 75%IPA
75%IP 75%IP 75%IP 75%IP
A A A A
2,2'-Dio1- a:1 a:1.08 a: 1.17 a: 1.06 a: 1.14 a: 1.33 a: 1.34 a: 1.24 a: 1
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5,5',6,6',7,7',8,8'- k1:13.0 k,:17.9 k1:11.10 k1:8.80 k,:12.16 k,:12.3 k1:7.83
k,:13.54 k,:12.73
octahydro-1,1'- 3 2 10%IPA 10%IPA 10%IPA 3 10%IP 10%IPA 10%IPA
binaphthalene 10%IP 10%IP 10%IP A
A A A
1,2,3,4- a: 1 a: 1.30 a: 1.18 a:1.19 a:1.15 a: 1.22 a: 1.48 a: 1.21 a: 1.26
Tetrahydro-4- k1:11.5 k,:11.3 ki:18.68 k1:7.91 k1:12.93 k1:13.3 k1:23.0
k1:12.98 k1:9.61
(4 2 4 15%IPA 15%IPA 15%IPA 6 7 15%IPA 15%IPA
methoxyphenyl)-
6-methyl-2- 10%IP 15%IP 15%IP 15%IP
thioxo- A A A A
5-pyrimidine
carboxylic acid
ethyl ester
1,2,3,4- a: 1.07 a: 1.49 a: 1.20 a: 1.40 a: 1.36 a: 1.37 a: 1.26 a: 1.40 a:
1.36
Tetrahydro-4- k1:36.7 k1:39.0 k1:27.80 k1:23.11 k1:31.14 k1:27.1 k1:28.5
k1:24.64 k1:16.80
(4 2 0 50%IPA 30%IPA 30%IPA 8 6 40%IPA 30%IPA
hydroxyphenyl)-
6-methyl-2- 70%IP 30%IP 30%IP 40%IP
thioxo-5- A A A A
Pyrimidinecarbox
ylic acid ethyl
ester
1-[1,2,3,4- a: 1 a: 1.29 a: 1.20 a: 1.24 a: 1.11 a: 1.34 a:1 a: 1.34 a: 1.23
Tetrahydro-4- k1:26.5 k1:26.2 k1:40.60 k1:19.00 k1:32.93 k1:31.7 k1:50.8
k1:34.45 k1:17.30
(4 4 7 15%IPA 15%IPA 15%IPA 2 5 15%IPA 15%IPA
methoxyphenyl)-
6-methyl-2- 10%IP 15%IP 15%IP 15%IP
thioxo- A A A A
5-pyrimidinyl]
etlianone
Hexobarbital a:1 a:1 a:1 a:1.22 a:1.10 a:1 a:1 a:1 a:1
k1:29.9 kt:11.5 k1:22.28 k1:7.21 k1:13.11 k1:15.0 k1:9.74 k1:14.94 kl:15.27
2 1 1%IPA 1%IPA 1%IPA 2 2%IPA 1%IPA 1%IPA
1%IPA 1%IPA 1%IPA
Temazepam a: 1 a: 1 a: 1 a:1 a: 1 a: 1 a: 1 a: 1 a: 1
k1:20.7 kl:17.7 k1:25.54 kl:12.52 k1:17.34 k1:27.7 k1:25.4 k1:20.24 k1:27.30
7 3 2%IPA 2%IPA 2%IPA 5 4 2%IPA 2%IPA
2%IPA 2%IPA 2%IPA 10%IP
A
5-Methyl- a: 1 a: 1.30 a: 1.16 a:1 a:1 a:1 a: 1 a: 1.22 a: 1.16
5-(2,5-dichloro k1:9.35 k1:10.0 kl:14.6 k1:12.39 kl:15.78 k1:8.27 k1:7.52
kt:9.91 k1:7.30
phenyl)hydantoin 10%IP 0 15%IPA 15%IPA 15%IPA 15%IP 15%IP 15%IPA 15%IPA
A 15%IP A A
A
5-Methyl-5- a:1 a:1.18 a:1.10 a:1.08 a:1.16 a:1 a:1.16 a:1.10 a:1
phenyl k1:18.1 k1:17.9 k1:25.00 kl:12.93 kl:14.71 kt:13.5 k1:13.4 k1:19.00
kt:14.25
hydantoin 2 1 8%IPA 8%IPA 8%IPA 4 4 8%IPA 8%IPA
8%IPA 8%IPA 8%IPA 8%IPA
Mephenytoin a: 1 a: 1 a: 1.14 a:1 a:1 a:1 a: 1.10 a:1 a: 1.07
k1:7.42 k1:5.73 k1:7.86 k1:5.43 k1:5.43 k1:6.27 k1:9.74 k1:8.09 k1:9.00
2%IPA 2%IPA 2%IPA 2%IPA 2%IPA 2%IPA 2%IPA 2%IPA 2%IPA
sec-Butyl a:1.47 a:1 a:1 a:1.04 a:1 a:1 a:1 a:1 a:l
carbanilate k1:4.09 k1:9.04 k1:7.30 k1:5.91 k1:5.91 k1:7.74 k1:11.8 k1:9.64
k1:9.61
1%IPA 1%IPA 1%IPA 1%IPA 1%IPA 1%IPA 9 1%IPA 1%IPA
1%IPA
Methyl a:1 a:1.23 a:1.10 a:1 a:1 a:1.10 a:1 a:1 a:1
Mandelate k1:9.24 k1:9.00 k1:10.8 k1:6.50 kt:8.46 k1:6.27 k1:19.3 k1:19.00
k1:17.64
1%IPA 1%IPA 1%IPA 1%IPA 1%IPA 1%IPA 7 1%IPA 1%IPA
1%IPA
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Exanaple 6
This example compares chromatographic resolution of racemic compounds with
Fmoc-
Pro-Pro-Pro-Pro-N(Me)Ahx-APS (CSP-3) which is an embodiment of the present
invention, in
two mobile phase systems. Accordingly, this example helps demonstrate the
flexibility of chiral
stationary phases of the present invention in different mobile phase systems.
Table 3. Chromatographic resolution of racemic compounds with Fmoc-Pro-Pro-Pro-
Pro-
N(Me)Ahx-APS (CSP-3) in two mobile phase systems
Analyte name IPA/Hex DCM/Hex/MeOH
Benzoin a: 1.09 a: 1.07
k1:6.35 k1:11.61
3%IPA 5%DCM
in Hexane
Hydrobenzoin a:1.13 a:1.12
k1:17.98 k1:12.93
4%IPA 40%DCM
in Hexane
Benzoin oxime a: 1.13 a: 1.08
k1:15.36 k1:15.85
20%IPA 100%DCM
2,2,2-Trifluoro-l- a: 1.56 a: 1.20
(9-anthryl) k1:18.48 k1:9.54
ethanol 10%IPA 100%DCM
a-(pentafluoroethyl)- a: 1.10 a: 1.06
a-(trifluoromethyl)- k1:8.91 k1:28.23
Benzenemethanol 3%IPA 30%DCM
in Hexane
Warfarin a: 1.08 a:1
kt:11.19 k1:5.84
10%IPA & 30%DCM
1% in Hexane&
AcOH 1 %AcOH
Sec-Phenethyl a: 1.02 a: 1.02
alcohol k1:8.07 kl:13.08
1%IPA 10%DCM
in Hexane
a-Methyl-2- a:1.04 a:1
Naphthalenemethanol kt:17.62 kt:23.66
1%IPA 10%DCM
in Hexane
1-Acenaphthenol a: 1 a: I
k1:10.28 kt:7.31
3%IPA 30%DCM
in Hexane
3-Phenyl-Glycidol a.:1 a: 1
k1:6.77 k1:7.39
3%IPA 30%DCM
in Hexane
1,1'-Bi-2-naphthol a: 1.29 a: 1.06
k1:23.83 kl:12.21
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75%IPA 1 %MeOH
in Hexane
2,2'-Diol- a:1.32 a: 1
5,5',6,6',7,7',8,8'- k1:10.95 k1:12.08
octahydro-1,1'-binaphthalene 10%IPA 50%DCM
in Hexane
1,2,3,4-Tetrahydro4- a: 1.24 a:1.18
(4-methoxyphenyl)- kl:12.60 k1:6.26
6-methyl-2-thioxo- 15%IPA 60%DCM
5-pyrimidinecarboxylic acid
ethyl ester in Hexane
1,2,3,4-Tetrahydro-4- a: 1.41 a:1.19
(4-hydroxyphenyl)- k1:25.07 kl:12.72
6-methyl-2-thioxo-5- 30%IPA 3%MeOH
Pyrimidinecarboxylic acid
ethyl ester in Hexane
1-[1,2,3,4-Tetrahydro-4- a:1.21 a: 1.22
(4-methoxyphenyl)- kt:25.72 k1:12.20
6-methyl-2-thioxo- 15%IPA 60%DCM
5-pyrimidinyl]ethanone
in Hexane
Hexobarbital a:l a: 1
k1:16.98 k1:8.08
1 %IPA 30%DCM
in Hexane
Temazepam a: 1 a: 1
k1:20.24 k1:4.62
2%IPA 10%DCM
in Hexane
5-Methyl- a:1.17 a:1.12
5-(2,5-dichloro k1:8.55 kl:13.56
phenyl)hydantoin 15%IPA 100%DCM
5-Methyl-5-phenyl a: 1.15 a:1.11
hydantoin kl:15.6 kl:18.51
8%IPA\ 100%DCM
Mephenytoin a:1.14 a:1.17
k1:6.93 k1:17.10
2%IPA 20%DCM in Hexane
sec-Butyl carbanilate a: 1 a: 1.12
k1:7.64 k1:4.16
1%IPA 10%DCM in Hexane
Methyl Mandelate a: 1.17 a: 1
k1:8.31 k1:8.18
1 %IPA 1 0%DCM in Hexane
The invention being described, it will be apparent to those skilled in the art
that various
modifications and variations can be made in the present invention without
departing from the
scope or spirit of the invention. Other embodiments of the invention will be
apparent to those
skilled in the art from consideration of the specification and practice of the
invention disclosed
herein. It is intended that the Attachments be considered as exemplary only,
and not intended to
limit the scope and spirit of the invention.
19
CA 02571034 2006-12-14
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Unless otherwise indicated, all numbers expressing quantities of ingredients,
properties
such as molecular weight, reaction conditions, experimental results, and so
forth used in the
Specification and Attachments are to be understood as being modified by the
term "about."
Accordingly, unless specifically indicated to the contrary, are
approxiinations that may vary
depending upon the desired properties sought to be obtained by the present
invention.
References
The following references are incorporated by reference in their entirety.
(1) Stinson, S. C. Chemical & Engineering News 1995, 73, 44-74.
(2) Okamoto, Y.; Kawashima, M.; Hatada, K. Journal of the American Chemical
Society
1984, 106, 5357-5359.
(3) Yashima, E.; Yamamoto, C.; Okamoto, Y. Journal of the Ainerican Chemical
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1996, 118, 4036-4048.
(4) Berthod, A.; Chen, X.; Kullman, J. P.; Armstrong, D. W.; Gasparrini, F.;
D'Acquarica, I.;
Villani, C.; Carotti, A. Analytical Chemistry 2000, 72, 1767-1780.
(5) Ekborg-Ott, K. H.; Wang, X.; Armstrong, D. W. Microchetnical Journal 1999,
62, 26-49.
(6) Welch, C. J. Journal of Chromatography A 1994, 666, 3-26.
(7) Dobashi, A.; Dobashi, Y.; Kinoshita, K.; Hara, S. Analytical Chemistry
1988, 60, 1985-
1987.
(8) Billiot, E.; Warner, I. M. Analytical Chemistry 2000, 72, 1740-1748.
(9) Wang, Y.; Li, T. Analytical Chemistry 1999, 71, 4178-4182.
(10) Poole, C. F.; Poole, S. K. Chromatography today; Elsevier: New York,
1991.
(11) Creighton, T. E. Proteins. Structures and Molecular Properties. 2nd ed;
W. H. Freeman
and Company: New York, 1993.
(12) Carpino, L.; El-Faham, A.; Minor, C. A.; Albericio, F. Journal of the
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