Supercritical fluid chromatography system

Abstract

Provided is a chiller and system that may be utilized in a supercritical fluid chromatography method, wherein a non-polar solvent may replace a portion or all of a polar solvent for the purpose of separating or extracting desired sample molecules from a combined sample/solvent stream. The system may reduce the amount of polar solvent necessary for chromatographic separation and/or extraction of desired samples. The system may incorporate a supercritical fluid chiller, a supercritical fluid pressure-equalizing vessel and a supercritical fluid cyclonic separator. The supercritical fluid chiller allows for efficient and consistent pumping of liquid-phase gases employing off-the-shelf HPLC pumps. The pressure equalizing vessel allows the use of off-the-shelf HPLC column cartridges. The system may further incorporate the use of one or more disposable cartridges containing silica gel or other suitable medium. The system may also utilize an open loop cooling circuit using fluids with a positive Joule-Thompson coefficient.

Claims

1. A method of cooling via a circulator system utilizing the Joule-Thompson cooling effect of a fluid expanding through an expansion device located adjacent a pump head of a chiller pump to cool a refrigerant being pumped by the pump, the method comprising: (a) introducing the refrigerant into the circulatory system from a source container holding the refrigerant at ambient temperature, the system comprising an inlet portion, a pressurized portion and an expansion portion; (b) flowing the refrigerant from the inlet portion, comprising the source container connected, via a circuit to the pressurized portion comprising the chiller pump; (c) pumping into the pressurized portion of the system the refrigerant supplied from the inlet portion through the chiller pump with a pump head, the chiller pump pumping at a speed sufficient to keep the refrigerant in continuous circulation through the pressurized portion at: (1) a mass flow rate that is repeatable and proportionate to the operational speed of the chiller pump, (2) a continuous pressure of between 500 psi and 10,000 psi; (d) bringing the pressurized portion into fluid communication with a heat sink to allow heat to pass from a heated component external to the circulatory system, to the heat sink, and to the refrigerant circulating through the pressurized portion; (e) expanding a fluid in the expansion portion of the system through orifices of an expansion device located adjacent to the pump head of the chiller pump, the expansion device cooling the expanding fluid by virtue of the Joule-Thompson effect, the cooled fluid then cooling the pump head that in turn cools the refrigerant flowing into the pressurized portion from the chiller pump to a temperature between −5° C. and −30° C.

2. The method of claim 1, wherein the refrigerant is selected from the group consisting of hydrogen, nitrogen, argon, carbon dioxide.

3. The method of claim 1, wherein the fluid is the refrigerant.

4. The method of claim 1, wherein the fluid is the refrigerant and is supplied directly from the source container.

5. The method of claim 1, wherein the fluid is the refrigerant and is supplied from an outlet of the pressurized portion of the system.

6. The method of claim 1, wherein the refrigerant circulates through the system as a liquid and is maintained at a temperature that is warmer than the triple point temperature for the liquid.

7. The method of claim 1, wherein pressurized portion is configured to maintain a mass flow rate of between 10 milliliters per minute and 300 milliliters per minute of the refrigerant within the pressurized portion.

8. The method of claim 1, wherein pressurized portion is configured to maintain a mass flow rate of at least 50 milliliters per minute of the refrigerant within the pressurized portion.

9. The method of claim 1, wherein the system includes no more than one of said chiller pump.

10. The method of claim 1, wherein the system is configured to prevent the refrigerant from evaporating within the pressurized portion.

11. The method of claim 1, wherein the system is configured to prevent the refrigerant from forming condensate within the pressurized portion.

12. The method of claim 1, wherein the chiller pump is a piston-style positive displacement pump.

13. The method of claim 1, wherein the chiller pump is an HPLC—(High Pressure Liquid Chromatography-type) pump.

14. The method of claim 1, wherein the chiller pump is configured to pressurize the refrigerant within the pressurized portion to between 1,700 psi and 1,800 psi.

15. The method of claim 1, wherein the refrigerant within the pressurized portion is chilled at least 35° C. lower than the refrigerant in the source container.

16. The method of claim 1, wherein the expansion device contains at least one inlet orifice for fluid flow and at least one outlet orifice for fluid flow, and the expansion ratio between the at least one inlet orifice and the at least one outlet orifice is equal to or greater than 5 to 1.

17. The method of claim 1, wherein the refrigerant in the pressurized portion flows through a chromatographic column configured to allow the refrigerant to pass through a layer of stationary phase media to effectuate the separation of individual chemicals from a chemical mixture.

18. The method of claim 17, wherein internal and external pressure on the chromatographic column is balanced such that pressure differential on any wall separating the interior of the column from the exterior of the column is no greater than 200 psi.

19. The method of claim 5, wherein the system comprises an open loop cooling circuit configured to allow the fluid to be expelled from the circuit after passing through the expansion device.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a flow schematic of a chromatography system described herein.

(2) FIG. 2 illustrates an apparatus schematic of a chromatography system described herein.

(3) FIGS. 3A-B. FIG. 3A illustrates a block diagram of the chiller's two circuit cascade refrigeration system. FIG. 3B illustrates the chiller and HPLC pumps package. The post pump heater is used to bring process fluids up to operational temperatures.

(4) FIG. 4 illustrates a plot of mass flow rate vs. temperature at a set RPM of 250.

(5) FIG. 5 illustrates a plot of mass flow rate vs. temperature at a set RPM of 500.

(6) FIG. 6 illustrates a plot of mass flow rate vs. temperature at a set RPM of 750.

(7) FIG. 7 illustrates trend lines for mass flow vs. temperature at the set RPMs of 250, 500 and 750.

(8) FIG. 8 illustrates an assembly drawing of external views of a production prototype of the chiller. Enclosure (1). Power entry (3). Fan guard (23). Bulkhead union (25).

(9) FIG. 9 illustrates an assembly drawing of internal views of a production prototype of the chiller. Fan-cooled heat exchanger (2). Tube-in-tube heat exchanger (5). Sight glass; moisture indicator (12). Sight glass to service Tee Tube (14). Access valve (15). Service Tee to suction inlet tube (16). Capillary tube winding (21). Union elbow with tube fitting (22). Union coupling with tube fitting (24). Cryo compressor suction inlet tube (27). Liquid gas outlet tube (28). High capacity compressor drive (30). Mounting bracket (31). Drive board—compressor (32). 1.4 CC compressor (33).

(10) FIG. 10 illustrates an assembly drawing of internal views of a production prototype of the chiller. Fan mounting bracket (4). Reducing coupling (6). Compressor to condensing tube (7). Solder connection; copper fitting (8). 90 degree long elbow solder connection (9). Condenser to sight port tube (10). Copper tube, 45 degree elbow (13). 90 degree long elbow solder connection (17). Dryer, liquid line with service port (18). Down tube—condenser side (26). Liquid gas inlet tube (29). Power supply (34).

(11) FIG. 11 illustrates a cross sectional view of the pressure equalizing vessel showing the outer pressure containment vessel and the inner chromatography cartridge or column. Insets depict the input and output attachments of the inner chromatography cartridge or column to the outer pressure containment vessel.

(12) FIG. 12 illustrates a cross sectional view of the pressure containment vessel in the context of its fluid connections with an exemplary chromatography system.

(13) FIG. 13 illustrates an assembly drawing of the system, including a breakdown of the parts and quantities required.

(14) FIG. 14 illustrates a CFD visualization of the streamlines of the gas flow.

(15) FIG. 15 illustrates a manufacturing print of the Collection Cyclone Body.

(16) FIG. 16 illustrates a manufacturing print for the Collection Cyclone Cap.

(17) FIG. 17 illustrates a detailed internal geometry of the Collection Cyclone Assembly.

(18) FIG. 18 illustrates a separation of aceptophenone and methyl paraben using the chromatography system described herein.

(19) FIG. 19 illustrates a separation of aceptophenone and methyl paraben using the chromatography system described herein.

(20) FIG. 20 illustrates a separation of benzoic acid and 4-acetamidophenol using the chromatography system described herein.

(21) FIG. 21 illustrates a separation of ketoprofen and 4-acetamidophenol using the chromatography system described herein.

(22) FIG. 22 illustrates a schematic layout of a medium pressure flash chromatography system according to a preferred embodiment of the invention.

(23) FIG. 23 illustrates a cross sectional View of a pressure equalization system, i.e., pressure containment assembly according to a preferred embodiment of the invention.

(24) FIG. 24 illustrates a cooling system with an expansion device designed to impart fluid expansion to effectuate heat transfer where needed in the system.

(25) FIG. 25 illustrates an open loop cooling circuit for the refrigerant, wherein the refrigerant flows from a compressor through an inlet heat exchanger before passing to the first tube-in-tube heat exchanger.

(26) FIG. 26 shows a graph of exemplary refrigerants having a positive Joule Thomson coefficients.

(27) FIG. 27 shows a cross-sectional view of an exemplary primary heat exchanger incorporated into a pump.

DETAILED DESCRIPTION

1. Introduction

(28) Provided are supercritical fluid chromatography systems that enable the separation cartridges employed in traditional flash chromatography applications to be used in conjunction with a liquefied gas or supercritical fluid dominated solvent system. This facilitates or allows for a substantial reduction on the order 80-90% of the organic solvents or a complete elimination of organic solvents in the separation process. Achieving this goal involves implementation of one or more features to enable operating conditions in the range of pressures associated with subcritical fluids or subcritical fluids, e.g., in a pressure range of about 35 bar or higher pressure. These features include a prechiller system, a pressure equalizing vessel, and a pressurized cyclonic separator. When used in coordination, these improvements allow for liquefied gas and supercritical fluids to be utilized where only low pressure liquid solvents could previously been employed. The prechiller system enables a standard HPLC pump, nominally optimized for operation with incompressible fluids, to be employed with a liquefied gas or supercritical fluid. The pressure equalizing vessel enables an off-the-shelf chromatography cartridge, nominally intended for use with low pressure liquid solvents, to be used without further alteration in a liquefied gas or supercritical fluid system. The high pressure cyclonic separator enables product recovery from a high pressure system and serves the purpose of a collection flask in a high pressure system.

2. Supercritical Fluid Chromatography Systems

(29) The chromatography systems described herein are based, in part, on the discovery and advanced design of traditional flash chromatography technology that employs supercritical fluid (e.g., liquid phase CO.sub.2) as the main non-polar solvent in flash chromatography. FIGS. 1 and 2 illustrate a supercritical fluid CO.sub.2 flash chromatography apparatus. In varying embodiments, the apparatus has one or more of prechiller/supercritical fluid (e.g., CO.sub.2) pump that allows for the efficient and accurate delivery of liquid-phase supercritical fluid (e.g., CO.sub.2) in a supercritical fluid state to the apparatus up to 10,000 psi, e.g., up to 5000 psi or 2500 psi and at a flow rate of at least about 10 mls/min and up to about 250 ml/min or 300 ml/min, a secondary co-solvent pumping package that allows for polar modifier solvents (e.g., co-solvents) to be added to the flow stream in an isocratic or gradient mode (2500 psi and 100 mls/min), an injection manifold that can take the form of an injection loop or a secondary injection column for larger sample injection, a pressure equalizing vessel assembly that allows traditional flash column cartridges to be used in the apparatus up to the pressures of operation, a UV-VIS detector (other detectors optional), and a back pressure regulator (BPR) upstream of and in fluid communication with a stream selector, which is in fluid communication with the one or more cyclonic separators. The BPR brings the pressure of the flow stream from operation pressures down to ambient pressures for fraction collection in the cyclonic separators.

(30) Generally, the chromatography systems are pressurized to pump supercritical fluid (e.g., CO.sub.2), with or without co-solvent. In varying embodiments, the system further pumps a co-solvent. When pumping a supercritical fluid mixed with a co-solvent, the co-solvent may comprise up to about 20% v/v of the fluid being pumped through the system. As shown in FIG. 1, the co-solvent is delivered through an input pump separate from the supercritical fluid input pump, and mixed with the supercritical fluid prior to delivery to the inner column of the pressure equalizing vessel. In some embodiments, the co-solvent comprises an alcohol of 3 or fewer carbon atoms (e.g., methanol, ethanol, propanol, isopropanol) or an acetate of 3 or fewer carbon atoms (e.g., methyl acetate, ethyl acetate, propyl acetate), or mixtures thereof.

3. Chiller for Pumping Supercritical and Liquified Gases

a. Introduction

(31) A system for super chilling liquid gases (e.g., including carbon dioxide, methane, ethane, propane, butane, ethylene, propylene, and ethers) and to increase pumping efficiency and consistency is provided. The chiller cools liquid gases (e.g., including carbon dioxide, methane, ethane, propane, butane, ethylene, propylene, and ethers) to between −10° C. and −40° C. has been shown to enable the use of a standard HPLC pump with increased mass flow rates at a constant set point as the temperature is reduced. The herein described system reduces the cost of pumping CO.sub.2 by allowing the use of traditional HPLC pumps, rather than highly specialized CO.sub.2 pumps.

(32) The present systems and methods use a cascade chiller to cool the liquid CO.sub.2 to less than −10° C., e.g., in varying embodiments, less than −20° C., to minimize the variance in pump performance. The lower temperatures enable greater tolerance for the flow path in the high pressure CO.sub.2 pump head and facilitate the use of an unmodified HPLC (High Pressure Liquid Chromatography Pump). Traditional HPLC pumps are normally intended for pumping liquids; not liquid gases. There is a wide scatter in flow performance that results when the liquid is chilled to only 0° C. At room temperature conditions the variance would be greater than 30% and would render the unmodified HPLC pump completely ineffective in supercritical chromatography applications. The herein described chiller and methods allows the use of a traditional HPLC for precise metered pumping of liquid-gases, e.g., for delivery to extractors, reactors and chromatography equipment.

(33) We have determined that extreme subcooling much improved pumping performance. The pumping mass flow rate was linearly related to speed and repeatability. In this case, the supercritical CO.sub.2 was subcooled from an ambient condition of nearly 25° C. and 52 bar to approximately −25° C. and 52 bar. This 55° C. temperature reduction resulted in the liquid CO.sub.2 conditions more closely resembling an incompressible fluid, such as water. A completely unmodified and standard HPLC pump can then be used to pump the scCO.sub.2 under very linear conditions. Such behavior is highly desirable for applications including supercritical fluid extraction, supercritical fluid solid phase extraction, supercritical fluid flash chromatography, and supercritical fluid chromatography.

(34) The use of this hook of physics is advantageous in these applications, as it enables standard and cost-effective HPLC pumps to be used in supercritical fluid applications with highly linearly mass flow rates without the need for either elaborate compensation algorithms, sensor feedback systems involving compensation via a loss in weight measurement of the supply cylinder, direct compensation via a mass flow measure (e.g. coriolis mass meter), or the need for a booster pump to stabilize the delivery flow to the pumping system.

b. Prechiller or Chiller-HPLC Pump Assembly

(35) Generally, the prechiller or chiller utilizes dual refrigeration circuits with tube-in-tube heat exchangers that allow for heat exchange without an intervening heat exchange medium. FIG. 3A illustrates a block diagram of the two circuit cascade refrigeration heat exchanger present in the chiller. The two circuits are a low temperature circuit and a high temperature circuit. In such systems, the two circuits are thermally coupled at the condenser of the low temperature circuit. In fact, the condenser of the low temperature circuit is the evaporator of the high temperature circuit. To further simplify the concept, the low temperature circuit in the chiller is used to super chill the CO.sub.2 flow to its target temperature and the high temperature circuit is used to remove the heat from the low temperature circuit.

(36) CO.sub.2 flow enters the evaporator of the low temperature circuit at bottle pressure/temperature. Said evaporator is a tube in tube heat exchanger with an inner tube made of AISI Type 316 stainless steel or similar metal suitable for exposure to CO.sub.2. Other materials of use for the inner tube include without limitation copper, brass, and Type 304 stainless steel. Heat is removed from the CO.sub.2 by the flow of cryogenic refrigerant in the outside tube which is made of copper and surrounds the inside tube. The heat exchanger is set up as a counter flow heat exchanger for greater efficiency.

(37) The low temperature circuit is used to pull heat from the CO.sub.2 flow to chill it to the required temperature. A cryogenic refrigerant enters the suction side of the compressor and is discharged at a higher pressure. The compressor is a 1.4 CC model by Aspen. Work is done by the compressor to increase the pressure of the cryogenic refrigerant, which raises its temperature. The cryogenic refrigerant then exits the compressor on the discharge (high pressure) side and enters the low temperature circuit condenser. The low temperature circuit condenser is the same unit as the high temperature circuit evaporator. The condenser is a tube in tube heat exchanger. The cryogenic refrigerant flows through the inside tube of the heat exchanger. Heat is removed from the cryogenic refrigerant by a conventional refrigerant flowing in the outside tube which surrounds the inside tube. This heat exchanger is arranged as a counter flow heat exchanger for greater efficiency. After having the heat removed the cryogenic refrigerant flows through a moisture indicator, and then a dryer which has a built in service port. This is the high pressure side service port. After the dryer, the cryogenic refrigerant flows through an expansion valve. In this case, the expansion valve is a coiled length of capillary tube. When the cryogenic refrigerant exits the expansion valve, it is returned back to a low pressure state, which reduces the temperature before it enters the low temperature circuit evaporator. The cryogenic refrigerant flows through the outside tube of the evaporator and removes heat from the CO.sub.2 flowing through the inside tube which it surrounds. Upon exit, the cryogenic refrigerant flows through a moisture indicator and a service tee before returning to the suction side of the low temperature circuit compressor. This cycle is continuous.

(38) The high temperature circuit uses a similar flow path with one major difference. The condenser of the low temperature circuit is a fan cooled liquid to air heat exchanger. In the high temperature circuit, a conventional refrigerant enters the suction side of the compressor and is discharged at a higher pressure. The change in pressure is accompanied by a rise in temperature. The refrigerant then flows into the condenser where forced air is used to remove heat. This heat is transferred to the atmosphere and out of the system. The refrigerant then flows through a moisture indicator and a dryer with built in service port before going through the expansion valve. On exit of the expansion valve the refrigerant is returned to a lower pressure and thus lower temperature. The refrigerant then enters the evaporator. Here the refrigerant for the high temperature circuit absorbs heat from the low temperature circuit in a tube in tube heat exchanger. Upon exit the refrigerant goes through a service tee and a moisture indicator before returning to the suction side of the compressor. This cycle is continuous.

(39) To summarize the two circuit cascade system, it is easiest to follow the transfer of heat into and then out of the system. In the case of the chiller, heat is brought into the system by a stream of CO.sub.2. The removal of heat from the CO.sub.2 is the ultimate goal of the system. This heat is removed by the evaporator of the low temperature circuit. The low temperature circuit is then used to transfer heat to the high temperature circuit. This happens in the high temperature circuit evaporator which is also the low temperature circuit condenser. In the final stage of heat transfer, the high temperature circuit transfers heat out of the system and into the atmosphere in the high temperature circuit condenser. In short, heat cascades from the CO.sub.2 to the low temperature circuit then the high temperature circuit and finally the atmosphere.

(40) FIG. 3B illustrates the connection of the chiller to a traditional HPLC type SCF Pump with the addition of post-pump heaters to bring the fluids up to operational temperatures.

c. Embodiments of Prechiller or Chiller

(41) In varying embodiments, the chromatography system comprises a prechiller or chiller, as described herein, upstream of a pump to cool the supercritical fluid sufficiently such that it can be pumped through a standard off-the-shelf, commercially available flash chromatography or high performance liquid chromatography (HPLC) pump. The prechiller improves the pumping performance (e.g., the consistency) for a supercritical fluid, e.g., carbon dioxide such that system mass flow rates are proportionate to pump speed.

(42) In varying embodiments, the prechiller cools the liquid phase supercritical fluid (e.g., CO.sub.2) to a temperature of about −5° C. or less, e.g., −10° C., −15° C., −20° C., −25° C., or less, e.g., but above the triple point temperature, e.g., above about −55° C., e.g., to about −40° C., −45° C. or −50° C. Such supercooling or extreme subcooling reduced in much improved pumping performance. The pumping mass flow rate was linearly related to speed and repeatability. In varying embodiments, the prechiller subcools the supercritical fluid (e.g., CO.sub.2) from an ambient condition of nearly 25° C. to approximately −10° C. or lower temperatures. In varying embodiments, the system employs a 2-stage refrigerant on refrigerant chiller system to cool and liquefy the gas phase supercritical fluid. In varying embodiments, the system does not directly or separately cool the pump heads.

(43) This minimum of 35° C. temperature reduction resulted in the supercritical fluid (e.g., liquid phase CO.sub.2) conditions more closely resembling an incompressible fluid, such as water. A completely unmodified and standard HPLC pump can then be used to pump the supercritical fluid (e.g., liquid phase CO.sub.2) under very linear conditions. Such behavior is highly desirable for application including supercritical fluid extraction, supercritical fluid solid phase extraction, supercritical fluid flash chromatography, and supercritical fluid chromatography.

(44) The supercooling prechiller enables standard and cost effective HPLC pumps to be used in supercritical fluid applications with highly linearly mass flow rates with the need for either elaborate compensation algorithms, sensor feedback systems involving compensation via a loss in weight measurement of the supply cylinder, direct compensation via a mass flow measure (e.g., coriolis mass meter), or the need for a booster pump to stabilize the flow.

4. Pressure Equalizing Vessel

a. Introduction

(45) Pressure equalization assemblies and methods of use are provided. More specifically, provided is a pressure equalization assembly that enables the use of low-medium pressure columns for flash chromatography in a higher pressure supercritical fluid chromatography application. The pressure equalization assemblies allows the attachment of commercially available chromatography columns or cartridges to the cap of the vessel, e.g., via a luer lock fitting, and seals on the other end using an O-ring or gasket that is captured axially by the cap and tapered stem of said column. Sample stream pressure going through the column is balanced by external pressure applied to the same column to maintain a pressure differential that is less than the standard operating pressure of the column. In doing so, it ensures that the columns can be used without failure for high pressure supercritical fluid chromatography.

b. Embodiments of the Pressure Equalizing Vessel

(46) In varying embodiments, the supercritical fluid chromatography system comprises a pressure equalizing vessel. The pressure equalizing vessel is designed to allow the use of commercially available or off-the-shelf low to medium pressure columns (e.g., in the range of about 14-200 psi) traditionally used in flash chromatography at the higher pressures (in the range of about 1000 psi to about 10,000 psi, e.g., in the range of about 1500-2000 psi) used in Supercritical Flash Chromatography. The pressure equalizing vessels described herein allow the use of more economical pre-packed disposable columns in Supercritical Flash Chromatography, rather than expensive high pressure columns that must be re-packed by the user.

(47) The pressure equalization vessel described herein utilize pressure equalization to allow the low pressure columns to exceed their rated burst pressures. This is accomplished by pressurizing the outside of the column to a level that ensures that the pressure differential between the flow through the inside of the column and the equalizing pressure on the outside of the column remains within the rated pressure of the column. For example, if a column is rated at 200 psi normal operating pressure, and the user desired to run at higher pressure ranges of about 1000 psi to about 10,000 psi, e.g., 1500-2000 psi, the system would ensure that the equalizing pressure is within 200 psi of working pressure. Testing has proven this to be effective at preventing failure of the columns due to overpressure.

(48) The pressure equalization system allows the attachment of commercially available or off-the-shelf flash chromatography columns to the cap of the vessel via a luer lock fitting, and seals on the other end using an O-ring or gasket that is captured axially by the cap and tapered stem of said column. The pressure equalizing vessel is compatible for use with any commercially available pre-packed flash chromatography cartridge, including without limitation cartridges made by Grace (grace.com), Silicycle (silicycle.com), Biotage (biotage.com), Teledyne-ISCO (isco.com), Buchi (buchi.com), Interchim Inc. (interchiminc.com), and Agilent (agilent.com). The pressure equalizing vessel does not limit the size of the inner column cartridge that can be used, but is designed to adjust and accommodate to the chromatography cartridge appropriate for a desired separation. In varying embodiments, the inner column can contain in the range of from about 4 grams to about 350 grams stationary phase media, e.g., 4 grams, 8 grams, 12 grams, 20 grams, 80 grams, 120 grams or 330 grams stationary phase media. In varying embodiments, the inner column comprises a diameter in the range of about 0.5 inches to about 3.5 inches and a column length in the range from about 3.5 inches to about 11 inches. Illustrative diameter and length sizes of the inner column include without limitation 0.94 inches diameter×3.85 inches length (4 grams stationary media); 1.38 inches diameter×4.60 inches length (12 grams stationary media); 1.77 inches diameter×6.43 inches length (40 grams stationary media); 1.99 inches diameter×9.50 inches length (80 grams stationary media); 2.18 inches diameter×10.31 inches length (120 grams stationary media); or 3.39 inches diameter×10.55 inches length (330 grams stationary media).

(49) Sample stream pressure going through the column is balanced by external pressure applied to the same column to maintain a pressure differential that is less than the standard operating pressure of the column. In doing so, it ensures that the columns can be used without failure for high pressure supercritical fluid chromatography.

(50) FIG. 11 illustrates a cross sectional view of the system. It shows the pressure containment vessel, the medium pressure column, and the methods for attaching the column to the vessel itself. The fittings of the pressure equalizing vessel can be readily adjusted to accommodate the inner column being used, wherein the standard input fitting accommodates a female luer lock on the inner column and the standard output fitting accommodates a male slip fitting on the inner column. In the embodiment depicted in FIG. 11, a luer lock connection provided on the supercritical fluid (e.g., CO.sub.2) plus optional co-solvent inlet of the column seals the outside pressure from the sample stream pressure. The luer lock adapter is shown as a threaded adapter in this print, but may also be an integral machined part of the vessel cap, or also a welded adapter. On the other end of the column, the outside equalizing pressure, and the sample stream pressure are sealed from each other using an O-ring or gasket, on the outside of the column stem. The cap of the vessel has a shelf to capture said gasket and the column stem is tapered so that it also helps capture the gasket in position by providing an axial force. This tapered stem and the luer lock on the opposite end are typical of industry standard low-medium pressure columns.

(51) FIG. 11 also shows the inlet connection for the sample stream. This stream typically is composed of a supercritical fluid (e.g., CO.sub.2), optionally a co-solvent, and the sample to be separated. The fitting shown is a high pressure compression fitting made to seal on the outside diameter of appropriately sized high pressure tubing. The same type of fitting is used for the Sample stream outlet, and the pressure equalizing inlet. The pressure equalizing medium will typically be a supercritical fluid (e.g., CO.sub.2).

(52) FIG. 12 illustrates a cross sectional view of an example supercritical flash chromatography system with the pressure equalization system incorporated. FIG. 12 illustrates the typical inputs of supercritical fluid (e.g., CO.sub.2) and co-solvent, and shows how the system equalizes pressure in this case. Input flow of supercritical fluid (e.g., CO.sub.2) is split, one direction serves as the pressure equalizing fluid, and the other direction is used in conjunction with co-solvent to flow with the sample through the column. The system pressure is controlled by a back pressure regulator.

(53) The check valve after the input tee for supercritical fluid (e.g., CO.sub.2) ensures that the pressure is typically greater on the outside of the low pressure column. This means that if any leaks were to occur, the leaks would occur from outside equalizing fluid, into the column. This protects the valuable samples being separated from being lost.

5. Cyclonic Separator

a. Introduction

(54) In varying embodiments, the supercritical fluid chromatography system comprises a cyclonic separator. The cyclonic separator is designed to efficiently and effectively separate sample molecules from a liquid phase or gas phase stream of a supercritical fluid, e.g., CO.sub.2. The separator is designed to accept tangential input flow, e.g., via tube compression fitting, allowing the separator to accept typical industry standard tubing. Using a tangential inlet, the flow is channeled in a cyclonic flow around the separator to separate the molecules from the gaseous flow by centrifugal force. The separator deposits the sample molecules conveniently into an attached sample collection jar, and can be completely disassembled for complete cleaning. To ensure any molecules not successfully separated by the centrifugal forces of the cyclone are not released to atmosphere, a sintered filter of an appropriate size (e.g. having a porosity grade of G-5, or a pore size in the range of about 1-16 microns) can be pressed into the exit of the cyclone, allowing only the gaseous flow to escape.

b. Embodiments of the Cyclonic Separator

(55) The herein described cyclonic separators are designed to separate molecules from a gas phase supercritical fluid (e.g., CO.sub.2) flow and collect the molecules in a sample jar. In varying embodiments, separation procedures are performed at flow rates in the range of about 10-300 ml/min, e.g., about 250 ml/min, and at pressures in the range of about 1000-10,000 psi, e.g., about 1500-2000 psi or about 1,750 psi. The cyclonic separators can be used within a pressurized chromatography system and in fluid communication with a sample stream using compression fitting adapters and can be vented to atmosphere directly, or by hooking up a hose to the outlet. All materials of construction are suitable for use with corrosive solvents.

(56) Other forms of cyclonic separators have been used in the past to attempt to separate a desired sample from CO.sub.2/co-solvent streams in the supercritical fluid extraction products. These have been much cruder, simpler devices typically consisting of an inlet tube that would bring the fluid/particle stream into a collection vessel at 90 degrees, the product stream would circulate around the interior diameter of the collection vessel and the particulate products and modifier co-solvents would drop out and settle at the bottom of the collection assembly and the gaseous SCF CO.sub.2 would vent through and an outlet tube. The problem with these devices was always the loss of desired product to the fluid gaseous stream on the outlet. This was because none of the devices were designed to form a true cyclonic flow, nor were they equipped with proper filtration on the outlets. By contrast, with the presently described cyclonic separators, a cyclonic flow path is induced in which the gas is forced into rotational flow around the exit tube facilitated by the tangential inlet, and is then forced into a downward spiral in towards the low pressure region by the conical section. The low pressure region is in the middle of the volume where the exit is located.

(57) FIG. 13 illustrates an overall assembly drawing of the cyclonic separator and collection assembly. The cyclone body is connected to the fluid stream using a National Pipe Thread (NPT)×compression adapter. In this illustrated assembly, the compression fitting is sized for ⅛″ tube and the NPT fitting is 1/16″. Compression fittings sized in the range of about 1/16 inches to about ¼ inches find use. The cyclone cap threads into the top of the cyclone body and seals against an O-ring. This ensures that pressure is not lost through the threads. The cap has a sintered filter pressed into the exit to ensure that any sample molecules that may not have been separated by the vortex flow are captured and not released to atmosphere. Pore sizes of the sintered disc can be sized for particular compounds. In the illustrated iteration, sintered filter having a porosity grade G-5 is used (1-16 microns pore size). In varying embodiments, sintered filters with G-0 to G-5 porosity grade find use (G5=pore size in the range of about 1-16 microns; G4=pore size in the range of about 10-16; G3=pore size in the range of about 16-40 microns; G2=pore size in the range of about 40-100 microns; G1=100-160 microns; and G0=pore size in the range of about 160-250 microns).

(58) The cyclone body can be configured to be adapted to many standard collection jars. In the embodiment illustrated in FIG. 13, a 500 mL glass collection jar is used. The cyclone body can have a threaded bottom exit for attachment and sealing to the collection jar. The cap of the jar can have a through hole, which allows the cyclone body to be secured to the cap using a nut. This connection can be sealed using an O-ring, as illustrated.

(59) FIG. 14 illustrates the result of a Computational Fluid Dynamics (CFD) streamline study done to optimize the geometry of the cyclone at 250 mL/min @1,750 psi of supercritical CO.sub.2 flow. When the CO.sub.2 flow reaches the cyclone, it is no longer at such high pressures because the cyclone is open to atmospheric pressure. Because of this, the mass flow rate was calculated and then used to determine the velocity of the stream entering the cyclone. The shapes utilized have been done so to properly function with the parameters of the particular CFD program. Though the appearance may differ slightly from the assembly in FIG. 13, the internal geometry of the cyclone body is the same. The stream lines pictured illustrate the path and velocity of the fluid flow. The colors vary from red to blue, with red indicating the highest stream velocity, and blue indicating the lowest stream velocity. Most importantly, FIG. 14 illustrates the downward spiral, substantially non-overlapping stream lines typical of an optimized cyclonic separator.

(60) As illustrated in FIG. 14, flow enters the cyclone body tangential to the inside diameter. The flow then begins to rotate around the exit tube of the cyclone. The centrifugal forces exerted on the molecules in the stream lines send the molecules outwards to the wall of the cyclone body, where a boundary layer keeps the streamlines from recollecting the molecules. The molecules are then free to fall to the bottom of the collection assembly. As the stream lines travel to the bottom of the cyclone body and hit the conical section, the velocity slows and the pressure increases. This forces the streamlines up the exit tube which is a low pressure escape from the higher pressure conical section.

(61) FIG. 15 illustrates the manufacturing print released to the machine shop for the current revision of the cyclone body. All dimensions and information pertinent to the manufacturing of the part are present. In the illustrated embodiment, the threads used to secure the cap to the body are the 1″-24 Class 2-B threads. Thread size is determined by the body of the cyclone, wherein the thread size and conformation are selected to withstand pressure and secure the cap. Generally, the threads are larger than the inside diameter of the cyclone body. In the illustrated embodiment, the body is secured to the collection vessel using a ⅜″-32 National Extra Fine (NEF) thread. The function of the cyclone is not dependent on these threads, they were selected to aid in manufacturing and assembly.

(62) FIG. 16 illustrates configurations of the cyclone cap. In the illustrated embodiment, the cyclone cap is secured to the cyclone body via screw threads. The ledge at the top the cap allows for a sintered filter to be pressed into the cap.

(63) FIG. 17 illustrates the internal dimensions of the cyclone. These dimensions were honed by using CFD visualization of streamlines, as illustrated in FIG. 14. Dimensions important to the functionality include the ratio of the diameter of the outer circumference to the inner diameter of the mid-height of the funnel in the range of about 3 to about 4, e.g., about 3.5. In varying embodiments, the funnel has an angle in the range of about 30° to about 60°, e.g., in the range of about 35° to about 55°, e.g., an angle of about 30°, 35°, 40°, 45°, 50°, 55°, 60°. The illustrated embodiment depicts a 0.875 diameter to 0.250 diameter ratio (e.g., a ratio of 3.5) along with a 40 degree funnel angle. A further important dimension includes the dimensions of the protrusion at the bottom of the cap. Generally, the depth of protrusion at the bottom of the cap extends below the tangential inlet In varying embodiments, the depth of protrusion at the bottom of the cap extends, e.g., in the range of about 0.5 inches to about 1 inch, e.g., in the range of about, 0.6 inches to about 0.9 inches, e.g., about 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0 inches. This zone represents a part of the internal geometry of the cyclone collection assembly (see, FIG. 16).

6. Methods of Separating Molecules

(64) Further provided are methods of performing high pressure separation and/or extraction procedures using a flash chromatography system, comprising employing the chiller or prechiller, as described above and herein. The chromatography systems comprising a pressure equalizing vessel, as described herein, are useful for the separation of molecules that can be separated using liquid chromatography, e.g., flash chromatography employing commercially available off-the-shelf column cartridges and off-the-shelf HPLC positive displacement pumps. Generally, molecules that can be successfully separated when employing a supercritical fluid solvent have a higher density than the supercritical solvent, for example, the molecules may have a higher density than supercritical, liquid phase and/or gas phase supercritical fluid (e.g., CO.sub.2). In varying embodiments, the molecules to be separated in the presently described chromatography systems comprising a cyclonic separator are small organic compounds, peptides, polypeptides, lipids, carbohydrates, nucleic acids and/or polynucleotides. In varying embodiments, the molecules to be separated can have a molecular weight in the range of about 40 daltons (Da or 40 gram/mol) to about 1,000,000 Da (g/mol), or more, e.g., in the range of about 100 Da (g/mol) to about 10,000 Da (g/mol), e.g., in the range of about 100 Da (g/mol) to about 5,000 Da (g/mol).

(65) In varying embodiments, the methods entail inputting a sample to be separated that is dissolved or suspended in a supercritical fluid (e.g., CO.sub.2), with or without co-solvent, into the inner column of the pressure equalizing vessel assembly. In varying embodiments, separation procedures are performed at flow rates in the range of about 10-300 ml/min, e.g., about 250 ml/min, and at pressures in the range of about 1000-10,000 psi, e.g., about 1500-2000 psi or about 1,750 psi. The interspace of the pressure equalizing vessel surrounding the inner column is also filled with supercritical fluid at a pressure such that the pressure differential between the pressure within the interspace and the pressure within the inner space of the inside column is less than the pressure rating of the inner column (e.g., less than about 14-200 psi). Molecules in the sample are separated according to well-known principles of liquid chromatography using commercially available and off-the-shelf flash chromatography cartridges or columns packed with solid phase media commonly used in the art.

EXAMPLES

(66) The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

Mass Flow Rates Vs. Temperature Employing the Chiller

(67) FIG. 4 illustrates test data for mass flow rate vs. temperature at 250 rpm pump speed. The data is focused around the temperature of −10° C., where pumping consistency increases and mass flow rate and pumping efficiency also begin to increase with decreasing temperatures. At an average temperature of 0.3° C., average mass flow rate was 0.0433 kg/min with a standard deviation of 0.0024. At an average temperature of −7.95° C., average mass flow rate was 0.0447 kg/min with a standard deviation of 0.0030. At an average temperature of −14.75° C., average mass flow rate was 0.0487 kg/min with a standard deviation of 0.0018. At an average temperature of −23.53° C., average mass flow rate was 0.0525 kg/min with a standard deviation of 0.0004. At an average temperature of −30.78° C., average mass flow rate was 0.0546 kg/min with a standard deviation of 0.0005. All tests were performed at a constant RPM of 250, with a target pressure of 2,000 psi and set point flow rate of 13 mL/min. The data shows a trend of increasing mass flow rate below −10° C. and decreased variation.

(68) FIG. 5 illustrates test data for mass flow rate vs. temperature at 500 rpm pump speed. The data is focused around the temperature of −10° C., where pumping consistency increases and mass flow rate and pumping efficiency also begin to increase with decreasing temperatures. At an average temperature of 1.3° C., average mass flow rate was 0.0847 kg/min with a standard deviation of 0.0104. At an average temperature of −9.23° C., average mass flow rate was 0.1067 kg/min with a standard deviation of 0.0047. At an average temperature of −17.88° C., average mass flow rate was 0.1113 kg/min with a standard deviation of 0.0069. At an average temperature of −25.28° C., average mass flow rate was 0.125 kg/min with a standard deviation of 0.0005. At an average temperature of −30.37° C., average mass flow rate was 0.132 kg/min with a standard deviation of 0.0005. All tests were performed at a constant RPM of 500, with a target pressure of 2,000 psi and set point flow rate of 33 mL/min. The data shows a trend of increasing mass flow rate below −10° C. and decreased variation.

(69) FIG. 6 illustrates test data for mass flow rate vs. temperature at 750 rpm pump speed. The data is focused around the temperature of −10° C., where pumping consistency increases and mass flow rate and pumping efficiency also begin to increase with decreasing temperatures. At an average temperature of −0.13° C., average mass flow rate was 0.158 kg/min with a standard deviation of 0.003. At an average temperature of −8.1° C., average mass flow rate was 0.173 kg/min with a standard deviation of 0.0047. At an average temperature of −16.2° C., average mass flow rate was 0.181 kg/min with a standard deviation of 0.001. At an average temperature of −23.87° C., average mass flow rate was 0.192 kg/min with a standard deviation of 0.001. At an average temperature of −31.65° C., average mass flow rate was 0.201 kg/min with a standard deviation of 0.0004. All tests were performed at a constant RPM of 750, with a target pressure of 2,000 psi and set point flow rate of 70 mL/min. The data shows a trend of increasing mass flow rate below −10° C. and decreased variation.

Example 2

Separation of Aceptophenone and Methyl Paraben

(70) 0.1 grams of Aceptophenone and 0.1 grams of Methyl Paraben were dissolved in 2 mls of Ethyl Acetate. This sample was injected into the sample loop of the SCF CO.sub.2 Flash Chromatography unit with a flow rate of 50 mls/minute of SCF CO.sub.2 and 10 mls/min of Ethyl Acetate at 1750 psi (120 Bar) and 50° C. These materials were separated through the 40 gram silica cartridge column and collected in cyclonic separators with a 99%+ efficiency.

(71) The SCF CO.sub.2 Flash unit, for the purposes of the present and following examples, was operated at 50 mls/minute SCF CO.sub.2 and 10 mls/minute up to 17.5 mls/minute of modifier co-solvent in an isocratic or gradient mode at 1750 psi (120 bar) and 50° C. The SCF CO.sub.2 Flash Chromatography unit is capable of operation up to 2500 psi (175 bar) with a SCF CO.sub.2 flow rate of 250 mls/minute and co-solvent modifier flow rate of up to 100 mls/minute with a maximum operational temperature of 100° C. The Ultra-Chiller cools the CO.sub.2 liquid coming from the supply tank from ambient temperature down to −25° C. to −30° C. which allows for efficient and accurate pumping of the SCFCO.sub.2. Once the SCO.sub.2 liquid has been pumped, it flows through a pre heater that brings the fluid from the −25° C. to −30° C. pump exit temperature up to operation temperatures of up to 100° C. The fluid streams (a supercritical fluid, e.g., supercritical CO.sub.2, and Co-Solvent modifier) flow through a static mixer that ensures the homogeneous mixing of the fluids for delivery to the column assembly. Sample introduction into the unit occurs in two modes: samples dissolved in solvent up to 5 mls in size are introduced through a sample injection loop, larger samples can be introduced through a column injection manifold (reaction mixture is evaporated onto a course silica gel that is placed in the column assembly for injection).

(72) For the purposes of this work a 40 gram W. R. Grace traditional flash cartridge was used (Grace Reveleris Silica 40 micron, 40 gram, Lot #09071032, P/N 5146132, Pressure Rating 200 psi). However, the pressure equalizing vessel or Column Cartridge Containment Assembly can accommodate traditional flash cartridges from Grace (4 grams up to 330 grams in size) and flash cartridges from other flash chromatography vendors (Silicycle (silicycle.com), Biotage (biotage.com), Teledyne-ISCO (isco.com), Buchi (buchi.com), etc). The UV-Vis detector was set to 254 nm to detect the fractions coming from the separation column to then be collected in the Cyclonic Separator Assemblies. Each individual peak can be collected as a pure fraction. The results are shown in FIG. 18.

Example 3

Separation of Aceptophenone and Methyl Paraben

(73) 0.1 grams of Aceptophenone and 0.1 grams of Methyl Paraben were dissolved in 2 mls of Ethyl Acetate. This sample was injected into the sample loop of the SCF CO.sub.2 Flash Chromatography unit with a flow rate of 50 mls/minute of SCF CO.sub.2 and gradient of 10 mls/min to 17.5 mls/min of Ethyl Acetate at 1750 psi (120 Bar) and 50° C. These materials were separated through the 40 gram silica cartridge column and collected in cyclonic separators with a 99%+ efficiency. The results are shown in FIG. 19.

Example 4

Separation of Benzoic Acid and 4-Acetamidophenol

(74) 0.1 grams of benzoic acid and 0.1 grams of 4-acetamidophenol were dissolved in 2 mls of Methanol. This sample was injected into the sample loop of the SCF CO.sub.2 Flash Chromatography unit with a flow rate of 50 mls/minute of SCF CO.sub.2 and gradient of 10 mls/min to 17.5 mls/min of Methanol at 1750 psi (120 Bar) and 50° C. These materials were separated through the 40 gram silica cartridge column and collected in cyclonic separators with a 99%+ efficiency. The results are shown in FIG. 20.

Example 5

Separation of Ketoprofen and 4-Acetamidophenol

(75) 0.1 grams of ketoprofen and 0.1 grams of 4-acetamidophenol were dissolved in 2 mls of Methanol. This sample was injected into the sample loop of the SCF CO.sub.2 Flash Chromatography unit with a flow rate of 50 mls/minute of SCF CO.sub.2 and gradient of 10 mls/min to 17.5 mls/min of Methanol at 1750 psi (120 Bar) and 50° C. These materials were separated through the 40 gram silica cartridge column and collected in cyclonic separators with a 99%+ efficiency. The results are shown in FIG. 21.

(76) It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.