U.S. patent application number 11/899059 was filed with the patent office on 2008-03-06 for high pressure flash chromatography.
Invention is credited to Lalit Chordia, Todd Palcic, Harbaksh Sidhu, John Whelan.
Application Number | 20080053908 11/899059 |
Document ID | / |
Family ID | 39136650 |
Filed Date | 2008-03-06 |
United States Patent
Application |
20080053908 |
Kind Code |
A1 |
Chordia; Lalit ; et
al. |
March 6, 2008 |
High pressure flash chromatography
Abstract
A method for separating a sample using high pressure flash
chromatography is provided. The method comprises the steps of: i)
providing a pressurized vessel containing an adsorption material;
ii) pressurizing a compressible fluid, optionally containing a
cosolvent, to create a mobile phase; iii) premixing the sample with
the mobile phase or optionally placing the sample in the
pressurized vessel; iv) eluting the mobile phase through the
pressurized vessel, to obtain a separated sample; v) heating the
mobile phase containing the separated sample after the mobile phase
exits the pressurized vessel to remove the compressible fluid; and
iv) collecting the separated sample. The pressurized vessel
contains an adsorption material having a particle size of 10-100
microns, and the pressure of the adsorption vessel is held at
50-350 bar during elution. Also provided is an apparatus for
carrying out the above method.
Inventors: |
Chordia; Lalit; (Pittsburgh,
PA) ; Sidhu; Harbaksh; (Cranberry Township, PA)
; Palcic; Todd; (Pittsburgh, PA) ; Whelan;
John; (Pittsburgh, PA) |
Correspondence
Address: |
MEYER UNKOVIC & SCOTT LLP
1300 OLIVER BUILDING
PITTSBURGH
PA
15222
US
|
Family ID: |
39136650 |
Appl. No.: |
11/899059 |
Filed: |
September 4, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60841823 |
Sep 1, 2006 |
|
|
|
Current U.S.
Class: |
210/656 ;
210/198.2; 210/85 |
Current CPC
Class: |
B01D 15/165 20130101;
G01N 30/34 20130101; B01D 15/20 20130101; B01D 15/40 20130101; G01N
30/34 20130101 |
Class at
Publication: |
210/656 ;
210/198.2; 210/85 |
International
Class: |
B01D 15/08 20060101
B01D015/08 |
Claims
1. A high pressure flash chromatography apparatus comprising: a) a
pressurized vessel containing an adsorption material having a
particle size of 10-100 microns; b) a pumping means for
pressurizing and delivering a mobile phase to the pressurized
vessel, the mobile phase comprising a compressible fluid; c) a
loading means for loading a sample into the pressurized vessel and
onto the adsorption material; d) a pressure controlling means for
controlling the pressure of the mobile phase exiting the
pressurized vessel; e) a heat exchanger for controlling the
temperature of the mobile phase exiting said pressure controlling
means; f) a collection means for collecting fractions of the
sample; and g) a means for directing the mobile phase exiting the
heat exchanger to the collection means.
2. The apparatus of claim 1, further comprising a second pumping
means to deliver a cosolvent to the pressurized vessel.
3. The apparatus of claim 2, wherein the mobile phase delivered to
the pressurized vessel is between 10.degree. C. and 150.degree.
C.
4. The apparatus of claim 2, further comprising means for
controlling the flow of the cosolvent to the pressurized
vessel.
5. The apparatus of claim 2, further comprising a pumping means
after the pressurized vessel and before the collection means.
6. The apparatus of claim 2, further including a detection means
for detecting for detecting compound(s) of interest in the
sample.
7. The apparatus of claim 2, wherein the adsorption material is dry
packed in the pressurized vessel
8. The apparatus of claim 2, wherein said adsorption material is
piston packed.
9. The apparatus of claim 2, wherein the pressurized vessel
contains a separate cartridge containing the adsorption
material.
10. The apparatus of claim 2, wherein the adsorption material is a
silica-based material.
11. The apparatus of claim 10, wherein the silica-based material
has a particle size of between about 20 to about 70 microns
12. The apparatus of claim 2, wherein the mobile phase further
comprises a cosolvent.
13. The apparatus of claim 2, wherein said collection means is
selected from the group consisting of one or more fraction
collectors, cyclonic separators, trapping devices, solid phase
cartridges, cryofocusing systems and any combination of these.
14. The apparatus of claim 2, wherein said loading means is an
injection valve.
15. The apparatus of claim 2, wherein the loading means is an
injection pump in combination with an injection valve.
16. The apparatus of claim 2, wherein the loading means is manually
pouring the sample into the pressurized vessel.
17. The apparatus of claim 1 or 2 further comprising one or more
frits in the pressurized vessel.
18. The apparatus of claim 1 or 7, wherein said loading means is a
loading cavity in the pressurized vessel or optionally in the
cartridge.
19. The apparatus of claim 2 further comprising a recycling system
placed after said collection means to recycle the compressible
fluid.
20. A method for separating a sample using high pressure flash
chromatography, the method comprising the steps of: i) providing a
pressurized vessel containing an adsorption material; ii)
pressurizing a compressible fluid, optionally containing a
cosolvent, to create a mobile phase; iii) eluting the mobile phase
and the sample through the pressurized vessel, to obtain a
separated sample; iv) heating the mobile phase containing the
separated sample after the mobile phase exits the pressurized
vessel to facilitate the separation of the compressible fluid; and
v) collecting the separated sample, wherein the pressurized vessel
contains an adsorption material having a particle size of 10-100
microns, and the pressure of the adsorption vessel is held at
50-350 bar during elution.
21. The method of claim 20, wherein the mobile phase is heated
before entering the pressurized vessel.
22. The method of claim 20, wherein the adsorption material is dry
packed into the pressurized vessel.
23. The method of claim 20, wherein the adsorption material is
piston packed into the pressurized vessel.
24. The method of claim 20, wherein the adsorption material is
present in a cartridge within the pressurized vessel.
25. The method of claim 20, wherein the sample is loaded directly
onto the cartridge within the pressurized vessel.
26. The method of claim 20, wherein the sample is premixed with the
mobile phase prior to elution through the pressurized vessel.
27. The method of claim 20, wherein the mobile phase is recycled
after collecting the separated sample.
28. The method of claim 20, wherein the compressible fluid is
carbon dioxide.
29. The method of claim 20, wherein the cosolvent is an organic
solvent.
30. The method of claim 20, wherein said adsorption material is a
silica-based material.
31. The method of claim 20, wherein the mobile phase comprises a
compressible fluid and a cosolvent.
32. The method of claim 20, wherein the sample is loaded into the
cosolvent prior to being mixed with the compressible fluid.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional patent
application Ser. No. 60/841,823, filed Sep. 1, 2006, the teachings
of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Chromatography is a technique used to, among other things,
separate component elements of a starting material. Within the
general field of chromatography, there are several types.
Supercritical fluid chromatography (SFC) is a high pressure,
reverse-phase method that typically operates above the critical
point of the mobile phase fluid, and offers significant speed
advantage and resolution over traditional techniques such as high
performance liquid chromatography (HPLC). SFC employs carbon
dioxide or another compressible fluid as a mobile phase, sometimes
with a co-solvent, to perform a chromatographic separation. SFC has
a wide range of applicability and typically uses small particle
sizes of 3-20 microns for column packing material and is for
analytical to preparative scale applications because of the lower
pressure drop. In HPLC applications pressure at the top of the
column typically reaches up to 1000 psi but pressure at the bottom
is reduced to ambient pressure, creating a significant pressure
drop.
[0003] Liquid chromatography (LC) applies to a cruder, lower
pressure, lower performance technique for simple separations. Flash
chromatography is a form of adsorptive chromatography and is subset
of LC that uses a very simple, porous stationary phase with
particle sizes nearer to 100 microns often in a disposable
cartridge, or column. Because the particles in the packing material
are larger and often irregular, the columns are much cheaper and
are considered disposable. Pressure at the top of the column in
flash chromatography applications is typically up to 100 psi and
dropping down to ambient at the bottom of the column. Still (U.S.
Pat. No. 4,293,422) describes a method of adsorptive chromatography
in which the mobile phase is first admitted into a space above an
adsorbent bed of silica gel, then pushed through the bed with gas
pressure. Once the space is cleared, the mobile phase with
dissolved compounds for analysis is admitted, and it too is pushed
into the bed, displacing the earlier charge of neat mobile phase.
Then in a third step, a second charge of neat mobile phase forces
the solution through the bed, causing fractionation of the solute.
A subsequent disclosure by Andrews (U.S. Pat. No. 4,591,442)
describes a similar device, the main difference being in the
placement of the liquid holding space. Both disclosures focus on
mechanical design and methods for achieving flash chromatography at
relatively low pressure. More recently, Ritacco (US App.
2003/0102266) describes a convenient polymer-encased cartridge for
use as a single ended flash chromatography column. Anzar
(WO/2004-051257, US App. 2005/0287062) describes another type of
pre-filled cartridge for flash chromatography. Common features of
all of these disclosures are (1) an emphasis on instrumental
convenience, and (2) the use of an adsorptive bed that allows for
fast, although imprecise, separation of solutes. The disclosures
also emphasize gas and liquid chromatography applications of low to
moderate pressure.
[0004] The majority of all separations in flash chromatography use
a normal phase technique with solvents such as methanol, ethanol,
hexane, and heptane and occasionally the reverse phase technique
with water and acetonitrile. Chemists buy thousands of flash
chromatography systems per year to use primarily as a simple,
repeatable normal phase purification technique. Because of the vast
number of flash chromatography systems in medicinal chemistry
laboratories in pharmaceutical research environments, users,
insurers, regulators and environmentalists are growing increasingly
concerned with the vast amount of toxic waste solvent generated at
these sites. Given the obvious problems associated with unsafe,
toxic, flammable solvents, a new simple, normal phase technique
must be found that is fast and uses less toxic solvents.
SUMMARY OF THE INVENTION
[0005] The present invention overcomes problems with issues with
flash chromatography by using a compressible fluid. In some
embodiments the compressible fluid may be recycled or vented, which
eliminates or reduces the cost of recycling waste solvent. Carbon
dioxide is a preferred compressible fluid because it is a
non-toxic, non-flammable, inexpensive, inert, non-toxic sustainable
and renewable resource.
[0006] Accordingly, in one aspect the present invention provides a
method for separating a sample using high pressure flash
chromatography, the method comprising the steps of: [0007] i)
providing a pressurized vessel containing an adsorption material;
[0008] ii) pressurizing a compressible fluid, optionally containing
a cosolvent, to create a mobile phase; [0009] iii) eluting the
mobile phase and the sample through the pressurized vessel, to
obtain a separated sample; [0010] iv) heating the mobile phase
containing the separated sample after the mobile phase exits the
pressurized vessel to facilitate the separation of the compressible
fluid; and [0011] v) collecting the separated sample, wherein the
pressurized vessel contains an adsorption material having a
particle size of 10-100 microns, and [0012] the pressure of the
adsorption vessel is held at 50-350 bar during elution. In another
aspect, the present invention provides a high pressure flash
chromatography apparatus comprising: [0013] a) a pressurized vessel
containing an adsorption material having a particle size of 10-100
microns; [0014] b) a pumping means for pressurizing and delivering
a mobile phase to the pressurized vessel, the mobile phase
comprising a compressible fluid; [0015] c) a loading means for
loading a sample into the pressurized vessel and onto the
adsorption material; [0016] d) a pressure controlling means for
controlling the pressure of the mobile phase exiting the
pressurized vessel; [0017] e) a heat exchanger for controlling the
temperature of the mobile phase exiting said pressure controlling
means; [0018] f) a collection means for collecting fractions of the
sample; and [0019] g) a means for directing the mobile phase
exiting the heat exchanger to the collection means.
[0020] These and other aspects of the invention will become more
readily apparent from the following drawings, detailed description
and appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] For the present invention to be easily understood and
readily practiced, the invention will now be described, for the
purposes of illustration and not limitation, in conjunction with
the following figures, wherein:
[0022] FIG. 1 is a schematic representation of a preferred
embodiment of the apparatus of the present invention.
[0023] FIG. 2 is a schematic representation of a preferred
embodiment of the pressurized vessel of the present invention.
[0024] FIG. 3 is a schematic representation of an alternative
embodiment for loading sample into the pressurized vessel.
[0025] FIG. 4 is a schematic representation of another alternative
embodiment for loading sample into the pressurized vessel.
DETAILED DESCRIPTION OF THE INVENTION
[0026] As used herein in the specification and claims, including as
used in the examples and unless otherwise expressly specified, all
numbers may be read as if prefaced by the word "about", even if the
term does not expressly appear. Also, any numerical range recited
herein is intended to include all sub-ranges subsumed therein
[0027] A compressible fluid is used as a mobile phase, to elute the
sample containing a compound(s) of interest. More than one
compressible fluid can be used, e.g., a mixture. Suitable
compressible fluids include, for example, carbon dioxide, water,
ammonia, nitrogen, nitrous oxide, methane, ethane, ethylene,
propane, butane, n-pentane, benzene, methanol, ethanol,
isopropanol, isobutanol, monofluoromethane, trifluoromethane,
dimethyl sulfoxide, acetonitrile, hydrofluorocarbons,
chlorotrifluoromethane, monofluoromethane, hexafluoroethane,
1,1-difluoroethylene, 1,2-difluoroethylene, toluene, pyridine,
cyclohexane, m-cresol, decalin, cyclohexanol, 0-xylene, tetralin,
aniline, acetylene, chlorotrifluorosilane, xenon, sulfur
hexafluoride, propane or a combination thereof.
[0028] A preferred compressible fluid is carbon dioxide, because it
is nontoxic, inexpensive and widely available, as long as the
sample requiring separation has some solubility in carbon
dioxide.
[0029] The mobile phase may also contain a cosolvent, such as an
organic solvent. A suitable solvent is chosen based on the polarity
of the materials being separated and to increase the solubility of
the sample in the compressible fluid. Preferably, the amount of
cosolvent is less than 50 wt. %, based on the weight of the
compressible fluid and cosolvent mixture combined, more preferably
less than 40%, less than 30%, less than 20%, or even less than 10%.
It is possible that no cosolvent will be required, although
typically at least a small amount is necessary, e.g., about 1-10%,
to improve solubility of the sample in the compressible fluid. One
skilled in the art can easily select a suitable solvent based on
the characteristics of the sample.
[0030] The mobile phase may be comprised of a single mobile phase,
or more than one mobile phase, e.g., two or more mobile phases,
such as three or four. The composition of the mobile phase or
phases is determined by the required solvent strength of the mobile
phase. Typically, the more polar the solvent mixture, the more
polar the compounds that are separated, as would be understood by
one skilled in the art. The compressible fluid and cosolvent can be
delivered to the pressurized vessel in a mixed stream or in
separate streams, according to the needs of the user.
[0031] In one embodiment a first mobile phase may be a weaker
solvent to elute non-polar entities, while a second or additional
mobile phase may be up to 100% of a stronger solvent to increase
the solvent strength of the mobile phase and elute polar
materials.
[0032] The mobile phase or phases may also include gradients. In a
preferred embodiment a gradient of the cosolvent is added to the
compressible fluid, e.g, the composition of the mobile phase
changes over time, over the course of the separation. A gradient,
instead of a fixed amount of solvent, may produce quicker or better
chromatographic separation because gradients may change the
chemical characteristics of the combined stream that elute
different compounds under different conditions.
[0033] The mobile phase is passed through a pressurized vessel
containing an adsorption material, the vessel being pressurized to
maintain the compressible fluid at the appropriate pressure. In one
embodiment, the sample is first loaded into the pressurized vessel
before the mobile phase is added, for example, if the sample is
very viscous. In another embodiment, the sample can be premixed
with the mobile phase, and the mixture is then loaded in the
pressurized vessel. In yet another embodiment, the sample is
dissolved in a solvent and introduced into the stream of the
cosolvent prior to mixing the compressible fluid with the
cosolvent. The solvent can be the same as or different from the
cosolvent used in the mobile phase. In another embodiment the
sample is injected into the mobile phase.
[0034] The present invention in various modes of operation results
in rapid equilibration which means that there is very little time
required between runs and the next injection can be almost
immediately. Unfortunately, in normal phase HPLC, there is
significant time spent equilibrating the column before the next run
is started.
[0035] Suitable chromatography adsorption materials include
silica-based materials, such as silica, silica gel or alumina of
regular or irregular shape, and other column packing materials
known to those skilled in the art of chromatography. A preferred
packing material is silica.
[0036] Typical packing material in standard flash chromatography
includes highly porous, irregular particles of sizes greater than
50 microns. Smaller particles can be used in the present invention
than in traditional LC, HPLC or flash chromatography because there
is a lower pressure drop from the top of the column to the bottom
of the column, resulting from a less viscous mobile phase with
higher diffusivities. Preferably, the particle size of the
adsorption material used in the present invention is between about
10-100 microns, more preferably between about 20-70 microns.
[0037] The pressurized vessel is a cylindrically shaped column made
from a material that is capable of withstanding high pressures,
such as stainless steel. The vessel can have either or both a top
and bottom cap and may be of single piece or multi-piece
construction. In one embodiment the adsorption material is dry
packed directly into the pressurized vessel. In another embodiment
the adsorption material is piston packed using dynamic axial
compression (DAC), which provides the capability of rapid packing
and unpacking of the adsorption material. In another embodiment a
separate cartridge or column containing the adsorption material,
which may be pre-packed, is inserted into the pressurized vessel.
The sample is then loaded into or onto the cartridge and the
pressurized vessel is used as support, including pressure
reinforcement, for the cartridge. The cartridge may be of a
disposable nature and made from any material common to flash
chromatography columns, including but not limited to all types of
plastic or other non-swelling materials. Furthermore, the cartridge
may contain column regeneration features. One or more frits may
also be included in the pressure vessel or cartridge. The diameter
of the cartridge may be of any size, but preferably between about 1
cm to 20 cm, more preferably between about 2 to 10 cm.
[0038] While the mobile phase passes through the pressurized vessel
(or flash cartridge within the vessel) containing the adsorption
material, back pressure is maintained using a manual or automated
back pressure regulator.
[0039] A detector is used to detect and separate concentrations of
the compound(s) of interest in the sample. Suitable detection
devices include, for example, Mass spectroscopy Detector, UV/VIS
detector, Evaporative Light Scattering Detector, Flame Ionization
detector, Fourier Transform Infrared Spectroscopy Detector,
Infrared Detector, or other similar devices known to one skilled in
the art. The detector can be placed either before or after the back
pressure regulator. The nature of the detection does not limit the
practice of the present invention.
[0040] After the mobile phase passes through the pressurized vessel
or flash cartridge and the back pressure regulator, it passes
through a heater. The heat from the heater makes sure that part of
the mobile phase that is not a liquid solvent is converted into a
gas. After the mobile phase passes through the heater, the desired
and undesired fractions of the sample are collected. Any standard
collection method/apparatus used in chromatographic separations can
be used. Suitable collection means include, but are not limited to,
fraction collectors and cyclonic separators. Also suitable are
trapping devices such as a solid phase cartridge or a cryofocusing
system. Undesired fractions are directed to waste collectors.
[0041] After the separation and prior to the transfer of the mobile
phase to collection, additional compressible fluid and/or solvent
can be added to the mobile phase. This typically occurs after the
mobile phase exits the back pressure regulator, and can be a manual
or automated addition, and is used to reduce the aerosolizing
effect on the separated sample. If the stream exiting the back
pressure regulator aerosolizes, the separated sample may drop out
and clog the lines to the collection means. In certain
applications, by adding additional fluid the amount of the
separated sample that is recovered is increased. The solvent can be
the same as or different from the original cosolvent used in the
mobile phase.
[0042] Also after the separation and prior to transfer to the
collection system, the mobile phase is heated to vaporize the
compressible fluid.
[0043] Operating parameters such as pressure and temperature depend
on the specific physical and chemical characteristics of the
compound of interest in the sample, and can be determined by one
skilled in the art. In a preferred embodiment of the present
invention, the mobile phase is maintained at a pressure of between
about 50 bar to 350 bar. More preferably, the pressure of the
mobile phase is maintained at a pressure of between about 70 to 150
bar at the top of the pressurized vessel or flash cartridge. The
pressure change across the pressurized vessel or flash cartridge
from the top to the bottom is preferably between about 1-100 bar
and more preferably between about 1-20 bar. Most preferably the
pressure change is between about 5-10 bar. Since the pressure of
the mobile phase is higher than in traditional LC or HPLC
techniques, the mobile phase is less viscous and has higher
diffusivities. A less viscous mobile phase translates into a
reduced pressure drop across the column or cartridge. A mobile
phase with higher diffusivities provides faster separations. By
using a compressible fluid such as carbon dioxide as the mobile
phase the present invention reduces or eliminates the use of
organic solvents used in flash chromatography. Additionally, the
mobile phase can be used by itself to accomplish pressure
equalization between the inside and the outside of the cartridge,
when a cartridge is used.
[0044] The temperature of the pressurized vessel is typically held
at between about 10.degree. C. to 150.degree. C., more preferably
ambient temperature to about 80.degree. C., the temperature
selection being based on the type of sample, the particle size of
adsorption material and other operating parameters.
[0045] There is no pure limitation on the flow rate of the mobile
phase. Rather the flow rate is limited by the pressure change
across the pressurized vessel or flash cartridge, which is a
function of the particle size of the adsorption material and the
flow rate. The method of the present invention is much faster than
conventional flash separation, taking between about 30 to 70% less
time than conventional flash methods. The flow rate is typically
related to the size of the column. For the same size column, the
delta pressure (change in pressure from the top of the column to
the bottom of the column) generated is an order of magnitude lower
as compared to running pure incompressible solvents. This means for
the same flow rate, the method of the present invention will be
about 4 to 10 times faster from the time of injection to the time
of collection than conventional flash chromatography, when all
other parameters such as flow rate, size of column, packing of
adsorption material in the column and particle size of the
adsorption material are otherwise the same.
[0046] FIG. 1 illustrates an embodiment of the present invention.
The compressed fluid supply 43 is opened to allow the compressed
fluid to flow through a pre cooling heat exchanger 36 which is
controlled by a water bath 37. The compressed fluid reaches the
compressible fluid pump 38 and is pumped through a dampener 39 and
into a mixing chamber 24. Types of compressible fluid pumps include
but are not limited to, reciprocating and syringe pumps. Any pump
of the present invention may also be a compressor. The cosolvent
supply 21 is first primed to remove bubbles through the cosolvent
pump 22 using a prime valve 23. After priming the cosolvent, pump
22 is activated and the cosolvent is pumped into the mixing chamber
24 where the cosolvent and compressible fluid are mixed to create
the mobile phase. The mobile phase goes through a second heat
exchanger 25 where it reaches its desired operating temperature,
which, as described above, depends on various other operating
parameters and the nature of the sample. A suitable operating
temperature can be determined by one skilled in the art. Isolation
valve 26 is used when changing the pressurized vessel 28,
cartridges in the pressurized vessel 28 or when samples are loaded
directly into the pressurized vessel 28.
[0047] Sample introduction into the pressurized vessel 28 is
achieved one of several ways. In an embodiment shown in FIG. 1, the
sample solution 41 is introduced into the mobile phase by first
pumping it with the injection pump 40 into a sample loop, and then
the sample loop is brought into the mobile phase by the injection
valve 27. In another embodiment shown in FIG. 3, the sample is
introduced using a sample reservoir 41 with only an injection pump
40 and no injection valve. The sample in the mobile phase enters
the pressurized vessel 28 and continues on to the detector 29. In
both of these embodiments, the sample is introduced between the
isolation valve 26 and the pressurized vessel 28. In another
embodiment shown in FIG. 4, the sample is introduced between the
prime valve 23 and the mixing chamber 24.
[0048] After passing through the detector the mobile phase with the
sample exits a third valve 30 and the compressed fluid is vaporized
in the heat exchanger 31. Collection of the desired separated
sample(s) can either be done automatically through software
settings or manually. Collection valves 32 are activated at certain
times and the desired separated sample(s) continue into the sample
collectors 33 and then into collection containers 34. When there is
no desired separated sample in the mixed mobile phase the mixed
mobile phase goes to a waste collector 42 and then into the
collection containers 34. To keep nominal pressure on the sample
collectors 33 a back pressure valve 35 is used and then the exhaust
gas goes to a vent 44. The back pressure valve 35 may be manual or
automated, and the exhaust gas may be recycled back to a precooling
heat exchanger 36 instead of being vented. Recycling can be
achieved by maintaining a higher collection pressure and in one
instance includes the following components in a recycling system:
1) cooling water bath 2) condenser, 3) and storage tank.
[0049] FIG. 2 is an enlargement of the pressurized vessel 28 shown
in FIG. 1, and illustrates a preferred embodiment of the
pressurized vessel 28, a vessel assembly 1. A separate flash
cartridge 6 is inserted into the vessel assembly 1, which has a top
cap (2) and a bottom cap 9. There is one seal 3 for each of top cap
2 and bottom cap 9 to seal it with the vessel assembly 1. The top
and the bottom of the cartridge 6 is held in place by two holders 4
that are connected (separately) to the top cap 2 and to the bottom
cap 9. There is another seal 5 to seal the cartridge to the holder.
The mobile phase enters the vessel assembly 1 through port 7 and
exits from port 8. Optionally, there is a sample loading cavity at
the top of the flash cartridge 10. In another preferred embodiment
the adsorption material is dry packed directly into the vessel
assembly 1 instead of using the separate flash cartridge 6 filled
with adsorption material.
EXAMPLES
[0050] For the purposes of the examples below, the following
experimental set up was used. The flash cartridge packed with
silica (Biotage with 60A Kp-Silica, 43-60 microns, and a
75.times.300 mm bed) was placed in the pressurized vessel (Thar 5 L
vessel) and the cap 2 to the vessel assembly is closed. The sample
to be separated was dissolved in a suitable cosolvent. The
compressible fluid pump 38 (Thar P-200) and the cosolvent pump
(Thar P-50) were started and allowed to reach the desired flow
rates. The pressure was maintained by a valve 30 to the desired
pressure. The mobile phase passed through a detector (Gilson UV
detector) and onto a valve (Thar ABPR 200) that controls the back
pressure on the flash cartridge. While passing through the
detector, a signal is relayed to the collection valves as to when
to start collection into the desired fractions. The mobile phase
with the separated sample passed through another heat exchanger
that vaporized the compressible fluid into a gaseous state to allow
the liquid to be more easily collected as fractions. The fractions
were collected in a collection means (Thar CS-1L). All analysis of
the fractions was conducted on a Thar SFC Method Station using an
AD-H column from Chiral Technologies. The fractions were dissolved
to create a 10 mL solution. The flow rate of the compressible fluid
(carbon dioxide) was 3.4 mL/min and 0.6 mL/min of co-solvent
(methanol). The amount of co-solvent was held constant at 15%. Back
pressure was maintained at 100 bar. A photo diode array detector
from Waters Corporation was used as the detection means with a
signal frequency of 245 nanometers to 300 nanometers. Temperature
was maintained at 40 C.
Example 1
[0051] 6.5 g of an Acetaminophen-Benzoic Acid sample solution was
dissolved in 100 ml of methanol at standard temperature and
pressure (STP). The total flow rate was set at 150 g/min with the
main compressed fluid being carbon dioxide, with a gradient of
5%-35% methanol over 10 minutes and thereafter it was maintained at
35%. The operating conditions were as follows: 1) back pressure on
the flash cartridge was maintained at 100 bar 2) temperature of the
mobile phase (carbon dioxide and methanol) was maintained at 35 C;
and 3) the heat exchanger was maintained at 35 C. Technically the
pressure in the vessel is the back pressure on the cartridge+the
delta pressure across the cartridge. Since the delta P is small,
the pressure in vessel is nearly the same as the back pressure. The
sample was introduced into the system using an injector loop and
230 mg of the sample was loaded. Two fractions from these
injections were collected. Using the Thar Superchrom software
fraction 1 was found to be 98% pure of Acetaminophen, and fraction
2 to be 99.9% pure of Benzoic Acid. Below are chromatograms
illustrating the separation.
Example 2
[0052] 3.5 g of a sample containing 1.3052 g of Acetaminophen and
2.1947 g of Benzoic Acid was dissolved in 50 ml of methanol at STP
and loaded directly onto a sample loading cavity at the top of the
flash cartridge. The pressurized vessel was then closed and carbon
dioxide, as the mobile phase, was introduced into the system and
allowed to equilibrate. Carbon dioxide was pumped at 150 g/min with
a gradient of 5%-35% methanol over 10 minutes, and thereafter it
was maintained at 35%. The operating conditions were as follows: 1)
back pressure was maintained at 100 bar; 2) temperature of the
mixed mobile phase (carbon dioxide and methanol) was maintained at
35 C; and 3) the heat exchanger was maintained at 35 C. Sample
fractions were collected. Using the Thar Superchrom software
fraction 1 found to be 99% pure Acetaminophen, and fraction 2 to be
99.9% pure Benzoic Acid. Below are chromatograms illustrating the
separation.
Example 3
[0053] 7.18869 g of a sample containing 2.85839 g of Acetaminophen
and 4.3303 g of Benzoic Acid was dissolved in methanol at STP.
Carbon dioxide was the compressible fluid and was pumped at 150
g/min. A mobile phase was created with a gradient of 5%-35%
methanol over 10 minutes, and thereafter it was maintained at 35%.
The operating conditions were as follows: 1) back pressure
maintained at 100 bar; 2) temperature of the mobile phase (carbon
dioxide and methanol) was maintained at 35 C; and 3) the heat
exchanger was maintained at 35 C. The sample was loaded into the
mobile phase using an injection pump through a tee immediately
before the column. 718 mg of sample was loaded onto the column.
Fractions from these injections were collected. Using the Thar
Superchrom software fraction 1 was found to be 86% pure
Acetaminophen, and fraction 2 to be 99.9% pure Benzoic Acid. Below
are chromatograms illustrating the separation.
Example 4
[0054] 2.90041 g of a sample containing 0.32197 g of Ketoprofen was
dissolved in methanol at STP. Carbon dioxide was the compressible
fluid and was pumped at 150 g/min. A mobile phase was created with
a gradient of 5%-35% methanol over 10 minutes, and thereafter it
was maintained at 35%. The operating conditions were as follows: 1)
back pressure was maintained at 100 bar; 2) temperature of the
mixed mobile phase (carbon dioxide and methanol) was maintained at
35 C; and 3) the heat exchanger was maintained at 35 C. The sample
was loaded using an injection loop. 101 mg of sample was injected
onto the flash cartridge through the injection loop. Fractions from
these injections were collected. Using the Thar Superchrom software
fraction 1 was found to have increased the concentration of
Ketoprofen to 30% pure. Below is a chromatograms illustrating the
purification.
[0055] Various elements of the present invention can be practiced
individually or in any combination thereof without any limitation.
All elements disclosed in the present disclosure can be practiced
within the context of various industries including but not limited
to, pharmaceuticals, fine chemicals, nutraceuticals, coatings, and
petrochemical industries.
* * * * *