Note: Descriptions are shown in the official language in which they were submitted.
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-1-
R,r~PIa METHOD FOR SEPARATION OF
Field of the Invention
T'ne invention relates to the field of high performance liquid chromatography
and more
patiicularly to the held of high performance liquid chrom~stogrdphy separation
of snsali
organic molecules.
;o Baci~rantttl of the Invention
I-high performance liquid chroma~ographY (I~LC) is commonly used for
analytical
cad preparative separations of biapolymers and other organic molecules. For
instance, the
individual cornponent5 within a complex organic reaction mixture may be
separated by
hIFLC. HPLC is performed in a pressure-resistant tuba containing a stationary
adsorbent
Is which is the packing material. A pressure mechanism exerts pressure an a
mobile phase
applied to one end of the column and moves it through the coiunui causing it
to exit the
opposite end of the column. A sample containing a mixture of compounds is
injected onto the
column through a samplo injection port. As the sample moves through the
packing material,
zhc various components of the sample adsorb to the paxldng material with
different affinities.
2o The components, therefore, can elute from the column separately under
appropriate
conditions. On a reverse phase HPLC colutt>n, the c4aipouuds within a sample
are separated
based vn hydrapliabicity.
I-~PLC analysis may be performed in isocratic or gradient mode. An isocratic
I3PLC
separation is one which is carried out under 3 constant eluant Composition. A
gradient 13PLC
?s separation is characterized by a gradual change in the percentage of two ar
more solvents
applied to the colimm over time, ?he chaaga in solvent often is controlled by
a mixing device
which rnixcs solvent A and solvent B to produce the HPLC solvent just prior to
its movement
through the column. The amount of tirnc over which the gradient is changed
from one
extreme to the opposite extreme is the gradient time.
3o Generally in gradient chromatography it is believed that increasing the
flowrate
andlor decreasing the gradient time results in a loss of resolution, that is
the ability of the
colurc~a to separate the components within the mixture into discrete eluant
fractions. ~Snyder,
L., et al., "Practical HPLC Method Development"' YYiley-Interscience
PuBlicatfon, (199'x.
CA 02318143 2000-07-m :ANI~NpE~ SHEET
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- 1 a-
. Rapid methods for ~.e preparation and isolation of potential drug candidates
usir~
automated ~ynth~etic organic chemistry techniques to create aombiaatorial
libraries represents
an important advance in drug discovery. Certain combinatorial libraries
encompass a series of
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-2-
compounds having common structural features but which differ in the rurnber or
type of
group attached to the main structure, Each conspouad within a combinatorial
library created
by parallel synthesis is a separate sample housed in a tube or well of a
microtitrc plate. once
the library is completed, each sample is subjected to quality control analysis
to confirm that
'~,e particular sample includes the desired library component at the requisite
purity. Gcnc:ally
this is accomplished by subjecting the samples to 1~LC with UV, evaporative
light scatter
detection (ESLD), or mass spectrometry detection; IR; NMR; or any other
appropz~.te
analytical techniques. Tf~e qualitative analysis of such combinatorial
libraries by conventional
HPLC requires on the average 5 to 20 min~.ites in order to separate various
compounds within
the sampie.
a problem encountered with prior art methods for separation of compounds in
combinatorial libraries using HPLC is the length of time required for
separation of each
cam_ple. Fzch sample of a combinatorial library produced by parallel synthesis
must be
analyzed separ&tely to determine if that sample houses the appropriate
compound andlor to
separate the compounds in the mixture. Each library includes thoe~sands of
samples each of
which require an average t~ttn time of 10 minutes. The amount of time required
to perform
separation on these samples may run. on the order of months using standard
equipment and
methodology.
Summary of the Ipvention
?g Tho present invention prnvidcs rapid methods far the analysis and
preparative isolation
of relatively simple synthetic mixtures containing a small ntunber of
reagents, the product(s)of
interest and a relatively small number of side products using HPLC. The
methods of the
invention reduce the HPLC analysis rem time par sample from an average of 5 -
20 minutes
shown in the prior art (YJeller, et al., Molecular piversity, (1997), 3:61-70)
to less than one
25 rrsinute without a meaning~.il loss of resolution. Tho imrention depends in
part upon the
discovery that'small organic mclZCUles could be separated on a full gradient
reverse phase
HPLC by ~tri~miri~~ The ~'~ vole of cluant applied. to the column, maximi2ang
the linear
flow velocity of the eluastt sad compressing the grsdicat time to resolve a
peak at least eve.~y 2
seconds. A full gradient is def ned as a change in the soivcnt B concentration
of at least SU%.
3o For exampic, if the initial concentration of solvent B was 15%, a full
gradient would be
achieved when the canccntrataon of solved B reached 6S%. TEZe prior art
believed that if the
total eIuarn volume was decreased and the flow rate increased to the levels
indicated in the
invention, the resolution of the peaks eluting off the column would be
AMEN~S~ SHEET
CA 02318143 2000-07-17
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WO 99/39195 PCT/US99/02371
-3
significantly decreased to an extent that it would not be possible to obtain a
discrete separation of
a mixture of small organic compounds.
The methods of the invention include applying a mixture of compounds to a
reverse
phase column configured in a gradient high performance liquid chromatography
system, and
operating with a flow rate of at least S column volumes/min. A complete
gradient is applied to
the column at a rate which uses a maximum total volume of 10 column volumes;
preferably 5
column volumes in order to maximize speed. These parameters allow each small
organic
component within the mixture of compounds to elute in a distinct fraction from
the column with
sufficient resolution which permits a peak production of at least 1 peak/2
seconds. A one minute
1 o analysis, using a peak production of 1 peak/2 seconds, would translate
into an analysis which
could baseline separate more than 30 individual peaks.
The amount of time that the complete separation requires depends on the
parameters used
in the separation, such as the length of the column and the amount of solvent
used. Preferably
the mixture of compounds is applied to the column at a first time point and
all the compounds are
I s eluted within a time period of less than one minute from the first time
point. In other preferred
embodiments all compounds are eluted within a time period of less than 30
seconds. In other
embodiments all compounds are eluted within a time period of less than 20
seconds.
In one embodiment of the invention the method also includes the step of
detecting at least
one of the compounds as it elutes from the column. In another embodiment the
method includes
2o the step of collecting at least one of the compounds in a distinct fraction
as it elutes from the
column.
In preferred embodiments, the mixture of molecules includes reactants and a
substantially
pure product of the reactants.
In other preferred embodiments, including those listed above, the column is
less than or
25 equal to 30 mm in length. The column is less than or equal to 15 mm in
length in other
embodiments.
According to other preferred embodiments, including those listed above, the
column has
a packing material which has an average diameter of less than 5 microns.
In other preferred embodiments, including those listed above, the peak
production is at
3o least 1 peak/ 1 second. The peak production is at least 1 peak/ 0.5 seconds
in other
embodiments.
Preferably the total volume of liquid applied to the column per analysis is
less than 15x
CA 02318143 2000-07-17
WO 99/39195 PCTNS99/02371
-4
column volumes, preferably less than 8x column volumes. The total volume of
liquid may
include a cleaning volume having a maximum of 2x column volume. In a preferred
embodiment
the. total volume of liquid may include an equilibration volume having a
maximum of 1 x column
volume.
In other embodiments, including those listed above, the mixture of molecules
is a
member of a combinatorial library of small organic molecules. Preferably the
combinatorial
library is made by means of parallel synthesis methods and the method is
performed for high
throughput purification and/or quality control analysis.
In additional embodiments the column flow rate has a linear velocity of at
least 3
1 o mm/sec. In another embodiment the linear velocity is 5 mm/sec.
The small organic compounds which elute from the column are typically analyzed
by a
detection device such as a UV detector. In one embodiment of the invention the
small organic
molecules are analyzed by both a UV detector and a mass spectrometer.
1n one embodiment the method is a method for analysis of at least one compound
in the
15 mixture of compounds. Preferably less than 10 ~g of the mixture of
compounds is applied to the
column for the analysis. In an embodiment the sample of compounds is not
collected for further
use after it is eluted from the column.
In another embodiment the method is a method for preparative isolation of at
least one
compound in the mixture of compounds. Preferably between 1 and 100 mg of the
mixture of
2o compounds is applied to the column for the analysis. In an embodiment the
compounds are
collected in separate fractions for further use after they are eluted from the
column. Preferably
the at least one compound is collected in a fraction having a volume of 2
milliliters or less.
The high performance liquid chromatography may be performed at a temperature
of
greater than 20 °C in one embodiment. In another embodiment the method
is performed at a
25 temperature of greater than 50 °C. In a preferred embodiment the
method is performed at a
temperature of greater than 60 °C.
In another aspect the invention is a rapid high performance liquid
chromatography
method for the preparative isolation of a concentrated fraction of a small
organic compound from
a mixture of compounds. The method includes the steps of applying the mixture
of compounds
30 to a reverse phase column in a gradient high performance liquid
chromatography system,
wherein the column has a flow rate of at least 5 column volumes/min, applying
a complete
gradient to the column in a maximum volume of l Ox column volume, causing the
small organic
CA 02318143 2000-07-17
WO 99/39195 PCTNS99/02371
-5
compound to elute in a distinct fraction, separate from the other molecules,
from the column such
that the elution permits resolution with a peak production of at least 1
peak/4 seconds and
collecting the small organic compound. The small organic compound, in some
embodiments is
collected in a fraction having a maximum volume of 2 milliliters.
In other embodiments, including those listed above, the mixture of molecules
is a
member of a combinatorial library of small organic molecules. Preferably the
combinatorial
library is made by means of parallel synthesis methods and the method is
performed for high
throughput purification and/or quality control analysis.
Each of the limitations of the invention can encompass various embodiments of
the
to invention. It is, therefore, anticipated that each of the limitations of
the invention involving any
one element or combinations of elements can be included in each method.
15 Figure 1 is a schematic representation of the instrumentation to perform
the method of the
presentinvention.
Figure 2 is a graph depicting the composition of the mobile phase during the
HPLC
analysis in terms of the number of column volumes of eluant applied to the
column.
Figure 3 is a chromatogram of a test mixture obtained using the method of the
present
20 invention with less than 1 minute total analysis time.
Figure 4 is a chromatogram of a test mixture obtained using the method of the
present
invention with less than'/Z minute total analysis time.
Figure 5 is a comparison of the chromatograms obtained for a sample
synthesized by
parallel synthesis using a 20 minute, a 1 minute, and a 30 second analysis.
25 Figure 6 is another example of a comparison of the chromatograms obtained
for a sample
synthesized by parallel synthesis using a 20 minute, a 1 minute, and a 30
second analysis.
Figure 7 is a series of chromatograms obtained using the method of the present
invention
in order to determine peak capacity obtained with various flow rates using a
50x4.6 m, 3 ~,m
Prontosil C 18-SH column: (7A) flow rate of 2 ml/min; (7B) flow rate of 3
ml/min; and (7C) flow
3o rate of 4 mUmin.
Figure 8 is a chromatogram obtained from a preparative isolation of l Omg of
each of the
CA 02318143 2000-07-17
WO 99/39195 PCT/US99102371
-6
three standard compounds using a 20 mm x 50 mm column packed with 5 um
particles and a
flow rate of 80 mUmin.
Figure 9 is a chromatogram obtained from a preparative isolation of
approximately 5 mg
of a mixture prepared by parallel synthesis using the conditions identified in
Figure 8.
Detailed Descriution of the Invention
The present invention provides new methods for the separation of small organic
molecules using reverse phase HPLC with applications for the analysis and/or
preparative
isolation of the separated compounds. Although the underlying principles of
analytical and
1 o preparative HPLC are the same (i.e., separating mixtures into discrete
components), traditionally
the mode of operation has been different. The goal of analytical HPLC has been
focused on
obtaining optimum resolution utilizing a minimum amount of material, whereas
preparative
chromatography has been focused on loading the maximum quantity of material
that could
satisfactorily be resolved. Moreover, since isolation of a particular
component is of interest in
15 preparative chromatography, the eluant is collected after separation. This
duality of operation
was reasonable when the analysis or preparative isolation was for a small
number of compounds,
where each analysis could be customized. However, using this same approach to
analyze and/or
purify a large number of different compounds synthesized by parallel synthesis
is too costly and
time consuming. In order to maintain a high throughput operation for both
analytical and
2o preparative isolation, new faster methods of the invention have been
developed where the
difference between analytical and preparative approaches are only in the scale
of the equipment.
By appropriately scaling up the column dimensions and the flow rate, the same
separation,
having the same resolution, can be achieved by loading 1 to 100 mg on a
preparative column as
can be achieved with 1 to 10 ug loading on an analytical column.
25 As used herein a "method for analysis" of small organic compounds is a
method in which
a sample of small organic compounds is loaded on a column and performed
according to the
methods of the invention described herein. In the method for analysis the
sample may or may
not be collected. In a preferred embodiment when the analysis method is
performed the sample
is not collected. Preferably the method for analysis involves the step of
loading less than 20 pg,
3o and even more preferably less than 10 pg, of sample on the column.
As used herein a "method for preparative isolation" of small organic compounds
is a
CA 02318143 2000-07-17
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method in which $ sample of small organic compounds is loaded on a column and
performed
according to the methods of the invention described herein and wherein the
compounds are
collected in fractions as they elute from the column. Preferably the method
far preparative
isola~aon involves the step of loading less than I00 mg, and even more
preferably between I
cad 100 zng, of sample on the column.
The new methods of the invention include both analytical and preparative
separations
and are signif cantly faster than prior art methods. The methods of the
invention are
particularly advantageous for separating components in small molecular weight
combinatorial
libraries as part of the quality control analysis andJor purific2~tion often
conducted for such
libraries. Prior tt~ the invention each sample of cer~.in combinatorial
libraries required
approximately S - 20 minutes for separation by HPLC' (Weller, et aL,
Molecaetur Diversity,
(199?), 3:61-70) .Using the rncthod of the invention, it has been discovered,
surprisingly, that
the separation time p~ sample can. be reduced to less than one mine~tc. 'ihc
time reduction
si~ificantly increases tha number of samples which can be separated per unit
time per
instrument. At a minimum, tlse iavert't'soa reduces the time of separation by
a factor of five
over prior art methods, enabling the separation of at least five times as many
compounds. In
preferred embodiments, the methods are more than 10 times faster than the
prior ~-t methods.
Using the prior art methods which typically require a total HPLO run time of I
O miwutes on
fully automated equipment, appro_5cimately 2,000 samples can be separated per
month. GTsing
2o the methods of the present invention, which only require a nxa time of 1
minute, 20,000
sFUnples can be separated in the same time period. The new methods are
described in detail
below.
Figure 1 illustrates the instntmernation useful according to the general
method of the
invention. 'Two solvent reservoirs, I2 and I~, housing solvents A and 8 are
pumped by
pumps 10 and IOA t~hrongh tubes 18a and I 8b, respectively, into mixing
chamber 16. A
computer 32 controls the amount of solvent A and B which is pumped into the
mixing
chamber over time. Solvents A and B are mixed iu the mixing chamber to form a
homogenous solvent which passes through tube 18c to a high pressure valve 19
and into a
column 20 containing a ravcrsc phase packing material. referred to as the
stationary phase. A
plurality of samples are housed within a microtitre plate 24 wlich rests in an
auto injector 22
which is also connected to the switching valve by tubing 18f. The sar~eple in
each well of the
plate 24 is firansported via. tubing 1$f through the switching valve and than
to the colxlmn
through tubing 18d by automated mans. Once the sample enters the column, the
CA 02318143 2000-07-17
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WO 99139195 PCT/US99/02371
_g_
the stationary phase with different affinities based on the hydrophobicity of
the compound. After
the sample is loaded the proportion of solvent A and solvent B is shifted with
respect to time in
order to create a gradient of solvent that is passed over the column. At
certain points within the
gradient, different small organic molecules are eluted from the column and
carried to a detection
device. The eluant is passed through a detection device such as a UV detector
28 to characterize
the compounds within the eluant. In some cases the eluant is exposed to
multiple detectors such
as a UV detector and a mass spectrometer 30. If isolation of a particular
component is of
interest, the eluant from the UV detector is split into a minor portion that
goes to the mass
spectrometer via tube 18g and a major portion which is transferred to the
fraction collector (31)
1o through tube 18i. The equipment necessary to practice the present invention
can be assembled
from commercially available devices. Although some of these devices were
actually designed
for other functions related to HPLC analysis, they can easily be adapted to
the functions
described herein. For instance the 50 ~1 mixing chamber which is shown in a
preferred
embodiment for mixing eluant in the analytical setup is actually a chamber
which is ordinarily
used for post column derivitization purposes. Additionally, the other devices
which are
assembled to produce the equipment are used according to the preferred
embodiment of the
invention and are adjusted as described in more detail below to produce the
equipment useful for
practicing the method of the invention.
The methods of the invention depend in part upon the discovery that small
organic
2o molecules could be analyzed and purified on a complete gradient reverse
phase HPLC by
minimizing the volume of liquid applied to the column, maximizing the linear
flow velocity and
compressing the gradient time to produce a peak production of at least 1
peak/2 seconds. Prior to
this invention, it was believed that manipulating these parameters beyond the
levels described in
the prior art would significantly decrease the resolution of the peaks eluting
off the column to an
extent that it would not be possible to obtain discrete separation of a
mixture of small organic
compounds. For instance, combinatorial libraries containing small organic
molecules are often
separated with gradient reverse phase HPLC using a run time of approximately
five to twenty
minutes for quality control analysis. It was believed, according to the prior
art, that reducing the
total run time by decreasing column volume and increasing linear flow velocity
would produce
an equivalent loss in resolution. Surprisingly it was discovered according to
the invention that
run time could be reduced by five fold over the minimum run time which has
been described in
CA 02318143 2000-07-17
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... .., ,.i..:r - .. . . ~U~H~- ', ... . . . . . ... __ _ _ _ _ _ , .. .:
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the prior art with only a minimum loss in resolution, based on peak capacity,
if the gradient
time is also decreased. Thi3 finding indicates that the methods of the
invention can produce
much faster separations with minimum reduction ofpcak capacity than those seen
in the prier
art. The parameter of peak, capacity is defined as the number of baseline
separated peaks that
will fit within the time that the gradient is changed from a low percent of
solvent B to a high
percent of solvent B. In order to normalize between different analyses, the
peak capacity
divided by the gradient time is defined as the "peak production", and
expressed is units of
peaks/seGOnd. ~JSing the metE~ods of the invention at least I peakl2 seconds
can be resolved.
Preferably I peakll second and even maare preferably 1 peak! 0.5 seconds are
resolved using
to the methods of the invention. Rcsclution is the ability to distingush
individual compounds
eluting from the column. Adequate resolution according tQ the inversion is the
ability to
resolve 1 peak every ~ seconds. As used herein, this means that the peak width
at baseline is
oa average not more than 2 seconds. A deterudnatioa of resolution using a
~cneasure of
baseline peak vridth is found in L.R. Snyder, J.J. Kirkland,1.L. Glajch,
"Pra.cticai 1-IPLC
~~iethad Development" Z"d Bdition, John Wiley 8c Sons, Inc., (199'7.
As will be apparent from Figure 1 and the description above, several variables
will
affeci the peak production capacity of the mctl:.od. Amongst these are (I )
size of the ooiumn,
(2) the packing n2atexial used in the column, (3) the use of a full gradient
in a minimum
volume, (4) the to'taI volume of liquid passing ovtr the column, and (S) the
linear flow
2o velocity. These variables should be adJusted, as further described below,
to produce a rapid
method haying a maximum run time of one .minute and a peak production of at
least 1 peakrl
seconds,
In preferred embodiments, the column used according to the m~~chod of the
invention
is short and wide. Prefexabiy the column has a length of less than 30 mm. A
shorter column
allows for a higher flow rate. With longer columns, flow rate roust be reduced
in order to
azininaize the back pressure which is created within the column. Additionally
a wide column,
such as a column having an internal diameter of greater than 4 mm, is
preferred in order to
minimize extra column band broadening associated with other parts of ta'~e
instrumentation.
However, a column having any width may be need according to the methods of the
invention.
3o A preferred width for analytical HPLC is between 4 and 5 mm. A preferred
width for
preparative isolation is between 20 and 30 mm.
The packing material used in the eoleunn is a solid support particle with
reverse phase
AMENDED SHEET
CA 02318143 2000-07-17
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WO 99/39195 PGT/US99/02371
-10
properties. Preferably, the packing material has a particle size of less than
5 ~,m and more
preferably less than 4 pm. Such packing materials are commercially available
and are well
known to those of skill in the art. It is possible that improved packing
materials will be
developed and in such case the preferred particle size may vary depending on
the improvement in
the materials.
The appropriate volume of solvent applied to the column is an important
parameter to the
method of the invention. Ordinarily, approximately 30 column volumes of
solvent is applied to a
column in a full gradient HPLC analysis and often more for preparative
isolation. The maximum
volume used according to the invention is 15 column volumes and preferably 8
column volumes
1 o to maximize speed. A graph depicting the maximum volume of liquid applied
according to the
methods of the invention in units of column volume is presented in Figure 2.
T'he volumes used
in prior art methods is presented in parentheses below the volume of the
invention. As shown in
the figure a complete gradient is applied to the column within 10 column
volumes over a time
course of 20-40 seconds. Prior art gradients require 15-30 column volumes to
achieve a
complete gradient. A cleaning cycle of 2 column volumes of 95% acetonitrile or
other elution
solvent is used to flush any remaining molecules from the column over a time
period of
approximately 5-10 seconds. The column is then equilibrated within 1 column
volume and
subjected to initial solvent conditions for one column volume over a total
time period of
approximately 5-15 seconds.
2o The preferred mixing volume of the solvent is the minimum volume that
permits a
homogenous mixture of solvent A and solvent B. The minimum volume is achieved
by utilizing
a small volume mixing chamber and minimum volume of tubing. An appropriate
mixing
chamber for the analytical embodiment has an internal volume of less than 250
~,l and preferably
less than 50 ~1. Although such small mixing chambers are not commercially
available, 50 ~l
post-column reaction chambers, which are commercially available can be used as
a mixing
chamber. In addition to selecting a mixing chamber having a minimum size, it
is preferred that
the mixing chamber be static. A static mixing chamber as used herein is a
chamber or column
packed with beads, usually made of steel or glass. As the solvent moves over
the beads, it is
subjected to turbulence and caused to be mixed together.
It is important that the method of the invention be performed using a complete
gradient in
order to assure that all components injected on the column have been removed
from the column
CA 02318143 2000-07-17
WO 99/39195 PCT/LTS99/02371
-11
prior to the injection of the next sample. A "complete gradient" as used
herein is a gradient of
solvent which begins with a low percentage of solvent B (solvent B is a non-
polar solvent such
as acetonitrile) and a high percentage of solvent A (solvent A is the aqueous
phase). A low
percentage of solvent B is preferably below 20%. The percentage of solvent A
and B is shifted
with time to produce a high percentage of solvent B and a low percentage of
solvent A. A high
percentage of solvent B is preferably above 70%.
The flow rate and the mixing volume dictate the time for the two solvents to
reach the
HPLC column, which in our analytical system is much less than 0.1 minutes.
Using a 50 pl
mixing chamber, minimal tubing, and a flow rate of 3-5 ml/min the movement of
the solvent
1 o from the pumps to the column would be complete in 2 - 6 seconds. Another
important parameter
is the linear flow velocity, the velocity with which the solvent moves through
the column. The
linear flow velocity is dependent on the flow rate and the internal diameter
of the column.
Preferably the linear velocity is greater than 3 mm/sec.
An additional parameter that can be used to increase peak capacity is
temperature. As
demonstrated in the examples below, when the temperature of the sample through
the continuous
liquid path is increased over 20 °C, the peak capacity is significantly
increased. When combined
with the high flow rates of the invention, the methods performed under high
temperatures
provide significant increases in peak capacity. Thus, although the methods of
the invention may
be performed at any temperature suitable for HPLC analysis, higher
temperatures may be
2o preferred to increase the peak capacity of the system. In some embodiments,
the temperature
may be a temperature greater than or equal to 20 °C, 30 °C, 40
°C, 50 °C or 60 °C. When
selecting an appropriate temperature for the HPLC method, one of ordinary
skill in the art will be
aware that some compounds may be unstable at higher temperatures. Those of
ordinary skill in
the art will be able to identify those compounds which are unstable at high
temperatures.
The method of the invention is useful for separating small organic compound
mixtures.
The mixture of compounds can include a number of small organic compounds of
unknown
composition having variations in hydrophobicity. The method of the invention
has the capability
to resolve at least 30 compounds that exhibit different hydrophobicity
characteristics, as
identified by the gradient composition at which each compound elutes, in one
minute or less.
3o Under conditions in which the resolution power of the method results in a
peak production of 1
peak/1 second, the method of the invention has the ability to resolve
approximately 60 such
CA 02318143 2000-07-17
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>: : .: ~ ; _::: : ~ ,' : : -: :~ . ~ : . ~~~~:: ::: :::::.:>:::,.:
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-12-
compounds in one minute.
Usually!, the mixture of compounds includes much less than the maximum number
of
compounds capable of being resolved by the system. For instance, a preferred
mixture of
compounds includes reactants and a substantially pure product of the
reactants. A mixttn~e of
compounds containing a "subs~tiahy pure product of the reactants" as used
herein is a
~~~ containing primarily the in""ended product, a small amount of uarcacted
starting
materials, as well as a few (preferably less than Sj side products in
significantly lower
quantity than the intended product. Such a mixture is achieved by using
reactants which arc
only capable of producing a limited number of products under the given
reaction conditions.
to In a prefczrcd embodiment of the invc~ion, the mixture of compounds is one
derived from the
preparation of a combinatorial library, A "combinatorial library of small
organic co~cnpounds"
is a collection of closely related analogs that differ from each other in one
or more points of
diversity and are synthesized by organic techniques using multi-step
processes.
Combinatorial librarir~s include a vast number of Small organic compounds,
some of !Which
1s may have important biological activity.
Qne type of combinatorial lib~raiy, which is preferred according to the
i.~venfiion, is
prepared by moans of parallel synthesis methods to produce a compound array. A
"compound
array" as used herein is a collection of compounds identifiable by their
spatial addresses in
Cartesian coordinates and arranged s~sch that each compound has a eoaunon
n~.oiecular core
2o and one or more variable structural diversity elements. 'floe compounds in
such a cvmgou:~d
array are produced in parallel in separate reaction vessels, with each
compound identif ed and
tracked by its spatial address. Regardless of the relative amount of starting
materials and
products that axe in each reaction vessel after reaction, the method of the
invention can be
used to analyze the extent. of reaction andlor isolate the components.
Examples o~ parallel
25 synthesis mixtures and parallel synthesis methods are provided in U.S.S.N.
08II 77,497, filed
January 5,1994 and its corresponding PCT published patent apphcattion
WQ95I18972,
published July 13, 1995 and U.S. Patent'1~1o. 5,712,171 granted January
X7,1998 and its
corresponding PCT published patent application W09fi~Z2529.
Once a serial of Small organic compound tnixtutes are developed, the mixtures
can be
3o separated and analyzed to datera~ine the product farmed and the extent of
reaction. Each
mixture of compounds represents a separate sample that is injected otrto an
HPLC colturin.
As shown in Figure 1 each sample is hdd within a well of a rnicrotitre plate
24 in an
autoSamplcr 2,2. The
AMENDED SHEET ~~~
CA 02318143 2000-07-17
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?..............'.... ~,...:....~~~"'~::....: ~:::::::.::_ .,
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WO 99/39195 PCT/US99/02371
-13
samples are loaded and injected manually or automatically using equipment
controlled by a
computer 32. The automatic loading and injection of the samples is preferred
because it enables
the continuous loading of samples at a rate which will not limit the overall
process of analysis.
Prior to loading the sample on the column the column is conditioned by flowing
through
it the intended mobile phase. As discussed above, the column is packed with a
non-polar
stationary phase on solid support particles, and can be obtained from a
variety of commercial
sources. HPLC columns can be obtained from a variety of commercial sources
such as
MACMOD (Chaddsford, PA). Once the column is conditioned, the system is
initiated by the
injection of the sample. As the small organic compounds contact the non-polar
packing material
1o each molecule is adsorbed to the packing material. The affinity with which
each compound
adsorbs to the packing material is dependent on the hydrophobicity of the
individual compound.
The injection of the sample defines time zero for the run.
A gradient is applied to the column immediately after the injection of the
sample in order
to elute the compounds bound therein in distinct fractions. A gradient HPLC
system includes
two reservoirs, 12 and 14, each containing a different polarity solvent which
are pumped through
a mixing chamber 16 and over the column 20 by means of a pump. In a preferred
embodiment,
solvent flow is maintained by a non-pulsating HPLC pump such as that available
as part of a
Shimadzu (Columbia, MD) HPLC system. A full gradient from 15% to 95% of
acetonitrile is
then applied to the column. The compounds in the mixture injected on the
column which are
2o polar have a greater amity for the initial composition of the eluant than
the stationary phase
and are thus eluted more rapidly than the more non-polar compounds which have
a greater
affinity for the hydrophobic stationary phase Solvents typically used for
gradients in reversed
phase HPLC generally include acetonitrile, methanol, isopropanol and propanol.
Modifiers are
typically added to the mobile phase, primarily to buffer the pH to a certain
narrow range, and
include a variety of acids and bases such as phosphoric acid, perfluorinated
carboxylic acids and
amines.
An HPLC compatible detector is used to detect the presence of small organic
compounds
as they are eluted from the column. A compatible detector is one which is
capable of detecting a
signal from a compound in an eluant and which produces a signal to indicate
the presence of that
3o compound. The detector should allow data acquisition at a rate of greater
than 10 points per
second and preferably greater than 20 points per second. HPLC compatible
detectors include,
CA 02318143 2000-07-17
WO 99/39195 PCT/US99/02371
- 14
but are not limited to, fluorescent, electrochemical, IR, NMR,
chemiluminescent, UV and mass
spectrometry. Preferably the HPLC compatible detector is a UV detector 28,
since commercially
available UV detectors are capable of achieving the required data acquisition
rate when the
settings are adjusted to achieve maximal values. Furthermore, UV detectors are
generally
applicable to a large and diverse number of chemical classes and a large
variety of mobile
phases.
In some cases, the HPLC compatible detector is both a UV detector 28 and a
mass
spectrometer 30. The use of both a UV detector and a mass spectrometer is
preferred because it
allows the methods of the invention to achieve, both purity (by UV) and
structural (by MS)
1 o information for each separated and detected component being eluted from
the column.
Moreover, the mass spectrometer can be used as a high specificity mufti-
dimensional detector to
provide general information on a class of compounds or specific information on
a particular
compound. These beneficial properties coupled with the inherent high
sensitivity of the mass
spectrometer make it one of the most desirable detectors to be coupled with
the separation power
of HPLC.
In the aspect of the invention described above the method may be used for
analysis, such
as quality control analysis and/or for preparative isolation of at least one
component of a mixture
of compounds. In this aspect of the invention one or more compounds may be
collected after
separation on the column in distinct fractions. The HPLC compatible detector
is used to identify
2o the presence of a compound in each fraction and the fractions are separated
into an acceptable
container such as a tube or a well of a microtitre plate.
In another aspect the invention is a rapid high performance liquid
chromatography
method for the preparative isolation of a concentrated fraction of a small
organic compound from
a mixture of compounds. Each of the parameters described above in relation to
the method of
separation are also applicable to this aspect of the invention. The method in
this aspect of the
invention differs, however, from the above method in that the separation of
molecules is
performed only for the purpose of separating compounds in a mixture into
distinct fractions and
the compounds are collected for future analysis or use. The method is
performed as described
above except that the elution permits resolution with a peak production of at
least 1 peak/4
3o seconds. In some embodiments the elution permits resolution with a peak
production of at least
1 peak/2 seconds.
CA 02318143 2000-07-17
WO 99139195 PCT/US99/02371
-15
Preferably the compounds are collected in fractions having a volume of 2
milliliters or
less. The methods of the invention accomplish the separation of compounds in
such a short time
that the total volume that each compound elutes in 2 milliliters or less. This
is advantageous
because the compound of interest is present in a fairly concentrated form as
opposed to the prior
art methods where the compound of interest is often eluted in a minimum volume
of 4 milliliters.
As described above, many variations on these particular examples are possible
and,
therefore, the examples are merely illustrative and not limiting of the
present invention.
1o Example 1: Gradient Reverse Phase HPLC Analysis of Standard Mixtures of
Small
Organic Molecules.
Equipment: All analytical separations were performed on a Shimadzu (Columbia,
MD)
HPLC System consisting of two LC-lOAS pumps, a SIL-l0A autosampler, a SCL-l0A
system
controller and a SPD-l0A UV-detector. The system was modified for high
performance
application in the following manner. A low volume static mixer from Supelco
(Bellefonte, PA)
with a 250 ul cartridge was used for fast and efficient high pressure mixing
of the eluents. In
combination with the short 30 mm columns a 50 ul mixing cartridge was used to
further
minimize gradient delay. The original bypass of the Shimadzu autoinjector was
removed to
eliminate band broadening effects. Injection was performed in the partially
filled loop mode,
2o injecting between 1 and 5 ul in a 50 ul loop. Connections between injector,
column and detector
were made with 0.007" tubing and the length kept at a minimum to prevent extra
column band
broadening. The original flow-cell of the UV detector was replaced with a semi-
micro version,
having a path length of 5 mm and a smaller total volume of 2.5 ul. The
detector response was set
to 1 to allow for detection of rapidly eluting, narrow peaks. Data acquisition
was performed by
the Chromperfect software from Justice Innovations (Palo Alto, CA) at a rate
of 20
points/second.
Chemicals: Water and acetonitrile were HPLC grade, dimethylsulfoxide was
A.C.S.
grade from Baker (Philipsburg, NJ). All other chemicals were the highest
available purity grade
from Aldrich (Milwaukee, WI). The HPLC eluents were prepared by adding 0.1 %
(v/v)
3o trifluoroacetic acid to water (solvent A) and acetonitrile (solvent B).
HPLC Columns: The Zorbax SB-C8 columns (SO mm x 4.6 mm and 30 mm x 4.6 mm
CA 02318143 2000-07-17
PCT1US99/02371
WO 99/39195
-16-
both packed with 3.5 ~.m particles; 150mm x 4.6 mm packed with 5 pm particles)
were obtained
from MACMOD (Chaddsford, PA). The Prontosil C18-SH columns (50 mm x 4.6 mm, 3
~m
particles) was a gift from Bischoff Analysentechnik (Leonberg, Germany).
An example of a large difference in compound hydrophobicity is represented by
our
standard test mixture, which consists of the following components:
acetamidophenol, 2-
hydroxydibenzofiuan and t-butylphenoxybenzaldehyde. In a reverse phase HPLC
separation, the
acetamidophenol elutes during the initial part of the gradient, the 2-
hydroxydibenzofuran elutes
in the middle of the gradient and the t-butylphenoxybenzaldehyde elutes at the
end of the
gradient. A mixture of the three compounds was prepared in dimethylsulfoxide
(DMSO). The
to mixture was then subjected to HPLC analysis according to the methods of the
invention under
the conditions described above:
The test mixture was first analyzed with a column packed with reverse phase
silica
having 3.5 pm particle size, at a flow rate of 3 ml/min in addition to each of
the above
conditions. The compounds were eluted with a gradient of 15-95% acetonitrile
applied to the
t 5 column in 0.7 min with 10 second hold and 5 second equilibration time. The
results are shown in
Figure 3. Three major peaks, representing each of the three components of the
test mixture were
resolved in less than 1 minute. Based on the baseline peak width of the middle
peak,
approximately 60 compounds with varying hydrophobicities could be baseline
resolved with this
method.
2o The same mixture of compounds was analyzed with a column packed with
reverse phase
silica having 3.5 pm particle size, at a flow rate of 5 ml/min in addition to
each of the above
conditions. The molecules were, again, eluted with a gradient of 15-95%
acetonitrile applied to
the column in 0.3 min with a 10 second hold and 5 second equilibration time.
The results are
shown in Figure 4. Once again based on the width of the middle peak,
approximately 30
25 compounds of varying hydrophobicities could be baseline resolved with this
30 second method.
Example 2: Gradient Reverse Phase HPLC Analysis of Combinatorial Library
Samples Obtained by Parallel Synthesis.
One sample from two different combinatorial libraries prepared by parallel
synthesis by
3o methods such as those disclosed in U.S.S.N. 08/177,497, filed January 5,
1994 and its
corresponding PCT published patent application W095/18972, published July 13,
1995 and U.S.
CA 02318143 2000-07-17
WO 99/39195 PCTNS99/02371
-17
Patent No. 5,712,171 granted January 27, 1998 and its corresponding PCT
published patent
application W096/22529 were chosen for analysis using the methods described in
Example 1.
The results for sample A1990702D9 analyzed using a traditional 20 minute
analysis are
compared to those obtained from the 1 minute and 30 second analysis in Figure
5. The 20
minute method used a Zorbax SB-C8 (150 x 4.6 mm) packed with S,um particles
and a full
gradient (15% - 95% solvent B) at 15 ml/min. Although the peak capacity is
less for the shorter
analyses, the resolution is more than adequate to determine purity, even in
such a complex
sample which would be the worst case scenario. Similarly, the results for
sample
AQ130QC48H5 using the 20 minute, 1 minute and 30 second analyses are given in
Figure 6.
to Once again, the 1 minute method provides the same purity information as the
longer 20 minute
analysis. Moreover, the 30 second analysis provides similar information.
Although the small
peak between the two major ones is partially merged with the major component,
there is still
adequate resolution to indicate the presence of the minor component. The
foregoing written
specification is considered to be sufficient to enable one skilled in the art
to practice the
invention. The present invention is not to be limited in scope by the
disclosed embodiments,
since these embodiments are intended merely as illustrative of particular
embodiments of the
invention as enabled herein and any methods that are functionally equivalent
are within the scope
of the invention. Indeed, various modifications of the invention in addition
to those shown and
described herein will become apparent to those skilled in the art from the
foregoing description
2o and fall within the scope of the appended claims.
Example 3: Effect of Flow Rate on the Rapid Gradient Reverse Phase HPLC
Analysis of the Invention.
In order to determine the effect of flow rate on the rapid analysis methods of
the
invention, the method was performed using various flow rates ranging from 2 to
4 ml/min. The
data is shown in Figure 7. In the case of the very steep 1 minute gradient, an
increase in the flow
rate from 2 ml/min (resulting in a gradient volume of 2.4 column volumes) to 4
ml/min (4.8
column volumes) increases the peak capacity from 29 to 48. The larger gradient
volume
achieved by increasing the flow rate also lowers the total analysis time as
indicated by the elution
of the last peak. The peak production rate in this case almost doubles with
the flow rate from
3o 0.47 to 0.8 peak/sec. The linear flow rate at 4 ml/min was 6.4 mm/second
and the back pressure
was about 250 bar which is acceptable for a routine application. Thus, columns
can be run at
CA 02318143 2000-07-17
WO 99/39195 PCT/US99/OZ371
-18
very high linear flow rates with steep gradients in order to obtain a high
peak capacity and peak
production rate.
Example 4: Gradient Reverse Phase Preparative HPLC Analysis of Standard
Mixture of Small Organic Molecules.
Equipment: Preparative separations were performed with a system of two Rainin
Dynamax SD-1 pumps (Woburn, MA), a Gilson-215 (Madison, WI) configured as both
an
autosampler and fraction collector, a Shimadzu (Colombia, MD) SPD-l0A UV
detector with a
preparative cell. Injection was performed in the partially filled loop mode
injecting 250 ul in a 5
to ml loop. Detection was at 254 nm and the detector response was set to 1 to
allow for detection of
rapidly eluting, narrow peaks. Data acquisition was performed by Unipoint
software from
Gilson (Madison, WI).
Chemicals: Water and acetonitrile were HPLC grade, dimethylsufoxide was A.C.S.
grade from Baker (Philipsburg, NJ). All other chemicals were the highest
available purity grade
from Aldrich (Milwaukee, WI). The HPLC eluents were prepared by adding 0.1 %
{v/v)
trifluoroacetic acid to water (solvent A) and acetonitrile (solvent B).
HPLC Column: YMC ODS-A C18, 20 mm x 50 mm packed with 5 um particles
obtained from YMC (Wilmington, NC).
The standard test mixture of acetamidophenol, 2-hydroxydibenzofuran, and t-
2o butylphenoxybenzaldehyde was prepared at 40 mg/ml of each component in
DMSO. The
mixture was then subjected to preparative HPLC analysis. The flow rate of the
mobile phase was
80 ml/min and the compounds were eluted with a gradient of 10%-95%
acetonitrile applied to
the column in 1 minute with a 20 second hold and a 40 second equilibration
time. The fraction
collector was set to collect the second and third peak. The results are shown
in Figure 8. Based
on the baseline peak width, approximately 30 compounds with varying
hydrophobicities could be
resolved and collected with this method.
Example 5: Gradient Reverse Phase Preparative HPLC Analysis of a
Combinatorial Library Sample.
3o One sample from a combinatorial library prepared by parallel synthesis by
methods such
as those disclosed in U.S.S.N. 08/177,497, filed 3anuary 5, 1994 and its
corresponding PCT
CA 02318143 2000-07-17
WO 99/39195 PCT/US99/02371
-19
published patent application W095/18972, published July 13, 1995 and U.S.
Patent No.
5,712,171 granted January 27, 1998 and its corresponding PCT published patent
application
W096/22529 were chosen for analysis using the method described in Example 4.
However, the
detection was at 217 nm and 400 ul of a 30mM solution of the sample was
injected. The fraction
collector was set to collect the expected product peak, which was identified
as the last peak in the
chromatogram. The results of sample A1990703D9 are shown in Figure 9.
Example 6: Effects of Temperature on Rapid Gradient Reverse Phase HPLC
Analysis.
1 o In order to determine the effect of temperature on the rapid analysis
methods of the
invention, the method was performed using various temperatures ranging from 20
°C to 60 °C
under conditions such as different flow rates. The data is presented in Table
I. As shown in the
table, the peak capacity increases significantly when the temperature is
raised from 20 to 60 °C
and this increase in peak capacity is even more pronounced when the system is
run at high flow
rates. As shown in the table, the peak capacity of a sample run on a 30mm
column at 4m1/min
using a 1 minute method with a 0.75 minute gradient, increased from 44 at 25
°C to 55 at 60 °C.
This correlates to a 25% increased based on a corresponding decrease in peak
width. In a similar
experiment in which the flow rate and temperature are increased from
4m1/minute to Sml/minute
and from 25 °C to 60 °C respectively, the peak capacity
increases from 44 to 60, which is a 35%
2o increase. In addition to the changes in peak capacity, the eluent viscosity
is decreased at higher
temperatures. Faster flow rates at comparable pressure drops may also be
accomplished at higher
temperatures. For instance, a decrease in pressure of 50% was observed when
the temperature
was raised from 20 to 60 °C. Additionally, a 5-10% increase in velocity
of eluent was observed,
indicated by a decrease in retention time of the compound.
TABLE 2
4.6 x 30 mm
column
peak-capacity 4ml/min SmUmin 4mUmin 6ml/min
25C 25C 60C 60C
1.00 minute method44 47 55 60
0.75 min gradient
0.75 minute method37 38 44 48
0.50 min gradient
CA 02318143 2000-07-17
ect. v . v my : t_YH-~91~~:NCHEI~ ()S : ~'7 _ J _ p : ~ g : 57 . 617 724 244 J
-~ +q 8 8 nr,.~.: ,~..-..-...,.:;.r. hr..,.:.
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13:::::.:'..:..:.::.::::;,.:-::.:.:.:.:...
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:......:::.:..:...:. .:...:.......... .....,...........
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a1 ' - . .;. . ... . . .~ :..::..::. . ... ......::.::.: -.:~:::
,::n h::: .;:: . ~-.~v..;;.;~ah.Cf,.:::::::::::~::~s:~:~vns~.:h.::::
:~:::n~:~.~::::w:::::::::.:~::n
........... . . :..s..........::...... ..::. . .. ...,..
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.::.'::'.;:.:.~~.~'i..'..~'.'~..-.::._.::~::: 'n.::"......<...h....... ...:.
r.... :.:.:t.. ... ................:....... .........
- 20 -
O.SO minute method ~0 ~- 34
2g
0.35 min gradient
.~.6 x 5bmm
column
peak-capacity 4 Z 4 60C~ s 6
1.00 minute method37 53 ~ 39
0.75 min ~adieat
0.75 minute method 46
0.50 min gradient
During the experimentation concerning the effects of temperature, it was
discovered
that the existing equipment could not effectively carry out the appropriate
temperature change
without some modification. Initially a coiuznn heater jacket was used in order
to regulate the
temperature, The calutnn is immersed in a circulating liquid which is
temperature controlle3.
The cluents were brought to the desired temperature through heat exchange
:.agillaries that
were immerstd in a system regulated by a thermostatt. It was fond, however,
that the
temperature of the eIuent dropped from approximately 50 'C to abot~, 28
°C between the
mixing device and the column at the high flow rates required for the rapid
HPLC analysis of
the invex~iion. A modif ed version of the equipment was asranbed by immersing
a manual
injector valve together wit,~t the heat exchanger and the column in a water
bath of the desired
temperature for the c~-perimental condition. This modification to the
equipment provided a
homogenous temperature distribution throughout the liquid Bath and enabled the
characterization oftetnperat~tre effects descrs'bed above. Those of ordinary
skill in the ant will
he able to modify the existing equipment to accomplish these effects based on
this teaching.
We claim:
CA 02318143 2000-07-m 'AMENDED SHEET
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