Patent Publication Number: US-6656354-B2

Title: Apparatus and method for preparative supercritical fluid chromatography

Description:
This application is a divisional of U.S. application Ser. No. 09/607,316 to Terry Berger, et al, entitled APPARATUS AND METHOD FOR PREPARATIVE SUPERCRITICAL FLUID CHROMATOGRAPHY, filed on Jun. 26, 2000, now U.S. Pat. No. 6,413,428 which claims the benefit of provisional U.S. application No. 60/154,038 to Terry Berger, et al, entitled PREPARATIVE SUPERCRITICAL FLUID CHROMATOGRAPHY filed on Sep. 16, 1999. 
    
    
     BACKGROUND OF THE INVENTION 
     A substantial need exists for industries to recover purified components of interest from samples containing simple or complex mixtures of components. Many technologies have been developed to meet this need. For dissolvable, nonvolatile components, the technology of choice has been liquid elution chromatography. 
     Analysts have several objectives in employing preparative elution chromatography. First, they wish to achieve the highest available purity of each component of interest. Second, they wish to recover the maximum amount of the components of interest. Third, they wish to process sequential, possibly unrelated samples as quickly as possible and without contamination from prior samples. Finally, it is frequently desirable to recover samples in a form that is rapidly convertible either to the pure, solvent-free component or to a solution of known composition which may or may not include the original collection solvent. 
     In the case of normal phase chromatography, where only organic solvents or mixtures are used as eluants, typical fraction volumes of tens to hundreds of milliliters are common. The fraction must then be evaporated over substantial time to recover the component residues of interest. In reversed phase chromatography, where mixtures of organic solvents and water are used as the elution mobile phase, a secondary problem arises. After removal of lower boiling solvents, recovered fractions must undergo a water removal step lasting from overnight to several days. Thus, availability of the recovered components of interest is delayed by hours or days, even after the separation process is complete. This latter problem can create a serious bottleneck in the entire purification process when enough samples are queued. 
     Where difficult separation conditions exist or separation speed is a requirement, a subset of elution chromatography, known as high performance liquid chromatography (HPLC), is preferred. This HPLC technique is used both as an analytical means to identify individual components and as a preparative means of purifying and collecting these components. 
     For analytical HPLC, samples with component levels in the nanogram to microgram range are typical. Preparative HPLC systems typically deal with microgram to multiple gram quantities of components per separation. Preparative HPLC systems also require a means to collect and store individual fractions. This is commonly performed, either manually or automatically, simply by diverting the system flow stream to a series of open containers. 
     Drawbacks exist to the current use of preparative HPLC. Elution periods ranging from several minutes to hours are necessary for each sample. Further, even in optimal conditions only a small fraction of the mobile phase contains components of interest. This can lead to very large volumes of waste mobile phase being generated in normal operation of the system. 
     An alternative separation technology called supercritical fluid chromatography (SFC) has advanced over the past decade. SFC uses highly compressible mobile phases, which typically employ carbon dioxide (CO2) as a principle component. In addition to CO2, the mobile phase frequently contains an organic solvent modifier, which adjusts the polarity of the mobile phase for optimum chromatographic performance. Since different components of a sample may require different levels of organic modifier to elute rapidly, a common technique is to continuously vary the mobile phase composition by linearly increasing the organic modifier content. This technique is called gradient elution. 
     SFC has been proven to have superior speed and resolving power compared to traditional HPLC for analytical applications. This results from the dramatically improved diffusion rates of solutes in SFC mobile phases compared to HPLC mobile phases. Separations have been accomplished as much as an order of magnitude faster using SFC instruments compared to HPLC instruments using the same chromatographic column. A key factor to optimizing SFC separations is the ability to independently control flow, density and composition of the mobile phase over the course of the separation. 
     SFC instruments used with gradient elution also reequillibrate much more rapidly than corresponding HPLC systems. As a result, they are ready for processing the next sample after a shorter period of time. A common gradient range for gradient SFC methods might occur in the range of 2% to 60% composition of the organic modifier. 
     It is worth noting that SFC instruments, while designed to operate in regions of temperature and pressure above the critical point of CO2, are typically not restricted from operation well below the critical point. In this lower region, especially when organic modifiers are used, chromatographic behavior remains superior to traditional HPLC and often cannot be distinguished from true supercritical operation. 
     In analytical SFC, once the separation has been performed and detected, the highly compressed mobile phase is directed through a decompression step to a flow stream. During decompression, the CO2 component of the mobile phase is allowed to expand dramatically and revert to the gas phase. The expansion and subsequent phase change of the CO2 tends to have a dramatic cooling effect on the waste stream components. If care is not taken, solid CO2, known as dry ice, may result and clog the waste stream. To prevent this occurrence, heat is typically added to the flow stream. At the low flow rates of typical analytical systems only a minor amount of heat is required. 
     While the CO2 component of the SFC mobile phase converts readily to a gaseous state, moderately heated liquid organic modifiers typically remain in a liquid phase. In general, dissolved samples carried through SFC system also remain dissolved in the liquid organic modifier phase. 
     The principle that simple decompression of the mobile phase in SFC separates the stream into two fractions has great importance with regard to use of the technique in a preparative manner. Removal of the gaseous CO2 phase, which constitutes 50% to 95% of the mobile phase during normal operation, greatly reduces the liquid collection volume for each component and thereby reduces the post-chromatographic processing necessary for recovery of separated components. 
     A second analytical purification technique similar to SFC is supercritical fluid extraction (SFE). Generally, in this technique, the goal is to separate one or more components of interest from a solid matrix. SFE is a bulk separation technique, which does not necessarily attempt to separate individually the components, extracted form the solid matrix. Typically, a secondary chromatographic step is required to determine individual components. Nevertheless, SFE shares the common goal with prep SFC of collecting and recovering dissolved components of interest from supercritical flow stream. As a result, a collection device suitable for preparative SFC should also be suitable for SFE techniques. 
     Expanding the technique of analytical SFC to allow preparative SFC requires several adaptations to the instrument. First the system requires increased flow capacity. Flows ranging from 20 ml/min to 200 ml/min are suitable for separation of multi-milligram up to gram quantities of materials. Also, a larger separation column is required. Finally, a collection system must be developed that will allow, at a minimum, collection of a single fraction of the flow stream which contains a substantially purified component of interest. In addition, there frequently exists a compelling economic incentive to allow multiple fraction collections from a single extracted sample. The modified system must also be able to be rapidly reinitialized either manually or automatically to allow subsequent sample injection followed by fraction collection. 
     Several commercial instances of preparative SFC instrumentation have been attempted which have employed different levels of technology to solve the problems of collection. A representative sampling of these products includes offerings from Gilson, Thar, Novasep, and ProChrome. However, no current implementation succeeds in providing high recovery, high purity, and low carryover from sample to sample. For example, one system may use the unsophisticated method of simply spraying the collection stream directly into a large bottle, which results in high sample loss, presumably due to aerosol formation. Another system uses a cyclonic separator to separate the two streams, but provides no rapid or automated means of washing the separators to prevent carryover. Such instruments are typically employed to separate large quantities of material by repetitive injection so that no sample-to-sample cleaning step is required. Other systems use a collection solvent to trap a sample fraction into a volume of special solvent in a collection container. This technique uses relatively large quantities of hazardous solvents to perform sample collection, is prone to sample fraction concentration losses or degradation, and possible matrix interferences exist between fractionated samples and collection solvent constituents. 
     An example of a SFC system is illustrated outside of the outlined section  10  in FIG.  1 . The schematic flow diagram is a packed-column supercritical fluid chromatography (SFC) system from initial modifier supply to a detector. The system has a carbon dioxide supply tank  200 , line chiller  220 , pump  202 , modifier tank  204  and pump  206 , dampener and pressure transducer  208 , leading to a mixing column  210 , connected to an injection valve  212  that is connected to at least one packed chromatography column  214 , and a detector  216 . 
     In a SFC system, liquefied compressed carbon dioxide gas is supplied from cylinders  200 . High pressure tubing  218  connects the carbon dioxide reservoir tank  200  to the carbon dioxide pump  202 . The tubing may be cooled  220  prior to connecting to the pump  202 . The system uses two HPLC-type reciprocating pumps  202 ,  206 . One pump  202  delivers carbon dioxide and the other pump  206  delivers modifier  204 , such as methanol. The carbon dioxide and modifier are combined, creating a mixture of modifier dissolved into the supercritical fluid. 
     The combined supercritical fluid is pumped at a controlled mass-flow rate from the mixing column  210  through transfer tubing to a fixed-loop injector  212  where the sample of interest is injected into the flow system. The sample combines with the compressed modifier fluid inside the injection valve  212  and discharges into at least one packed chromatography column  214 . After fractionation of the sample occurs in the columns  214 , the elution mixture passes from the column outlet into a detector  216 . 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a fraction collection device for supercritical fluid flow systems. 
     It is a further object of the present invention to provide a device that collects fractionated components of sample solutes into one or more collection containers. 
     The present invention relates to sample recovery after separation by supercritical fluid chromatography or supercritical fluid extraction, and improvements therein. 
     More specifically, the present invention relates to optimally separating a liquid phase, containing sample components of interest, from a much larger gaseous phase after the controlled expansion, or decompression, of a single chromatographic mobile phase from a high working pressure to a lower pressure where it is unstable. The controlled decompression causes a phase separation between liquid and gaseous phases while at the same time aerosol formation is strongly suppressed within the transfer tubing. 
     It is a further object of the present invention to provide a device and method to separate monophasic fluids that are mixtures of highly compressed or liquefied gasses and organic liquid modifiers into gaseous and liquid phases inside transfer tubing prior to collection of fractions of the liquid phase into one or more unique collection chambers. The collection of fractions of the liquid phase into collection chambers minimizes liquid solvent use and waste through efficient gas and liquid phase separation prior to entering collection chambers. The collection technique uses no additional solvents for collection of fractions. 
     This invention provides a cassette bank of multiple chambers to collect and store separated or extracted fractions. Each collection cassette includes one or more collection chambers, and each chamber can receive a purified liquid fraction. Each chamber may hold a removable sample collection liner. The collection liners may be individually removed, substituted, stored, cleaned and re-used, or discarded. One purpose of the collection liner is to provide a simplified means of transporting the collected liquid fraction from the cassette. A second purpose of the collection liner is to provide a means to eliminate cross-contamination of consecutive samples by providing an easily replaceable, uncontaminated liner in each collection chamber for each sample. 
     The present invention manually or automatically controls one or more valves and a sealing mechanism for collection chambers such that multiple liquid phase fractions from one sample may be collected into one or more chambers without mechanically adjusting the collection chamber seals. This method allows for rapid switching between collection chambers in the event of closely separated peaks in the chromatograghic flow stream. 
     It is a further object of the present invention to facilitate a manual or automatic reset of the collection system to allow consecutive samples to be processed in a rapid manner. Technical difficulties arise in the implementation of a collection system that satisfies all the analysts objectives stated above. The major problem centers around the tremendous expansion (typically 500-fold) of the pressurized liquid or supercritical CO2 fraction of the mobile phase that violently transforms into a gas at atmospheric pressure. This transition has four major negative effects with regard to liquid phase sample collection. 
     First, as mentioned above, the expanding CO2 causes a severe temperature drop that has the possibility of forming dry ice and clogging the system. Since flows of preparative SFC systems are much higher than corresponding analytical systems, considerable more heat must be added to compensate for the temperature drop. Care must be taken; however, not to allow the actual temperature to rise in the flow system since this may cause damage to thermally unstable compounds of interest. Higher organic modifier content reduces the severity of this problem, both by adding heat capacity and by dissolving the CO2, thereby preventing dry ice formation. 
     Second, as the CO2 expands, it rapidly loses any solvating power it had in the compressed state. If components of interest are largely dependent on the CO2 for solubility they will lose their primary means of transport through the flow system. Solid components will accumulate and eventually clog the flow path causing system failure. Again, the organic modifier component is an important factor here since the liquid will continue to solvate the components of interest and transport them to a collection device. Care must be taken not to introduce too much heat into the flow stream as to drive the organic modified also into the gas phase, otherwise its beneficial effect of transporting the solutes will be lost. 
     Third, it is beneficial to complete the transition from liquid to gaseous CO2 in as short a period as possible after the initial decompression stage. While in the liquid state, CO2 can disperse the organic modifier containing components of interest even when it is not dense enough to have any significant solvating power. This dispersion can have the effect of remixing components that had been efficiently separated by the SFC process prior to decompression. The faster the CO2 can be converted the less chromatographic degradation can occur. Two factors seem to predominate in controlling the ability to volatilize the liquid phase CO2: a) efficient heat transfer between the heat source and the flowing liquid and b) residence time of the CO2 in the heated region. The first factor can be positively affected by selection of a highly conductive material such as copper for heater fabrication. Insuring excellent thermal contact between the heater and a thin-walled transfer tubing also facilitates heat transfer to the flowing fluids. Residence time of the decompressing fluid can be controlled by stepping the pressure drop over a series of one or more restrictors in the transfer line. Higher backpressure slows the linear velocity of the biphasic fluid in the heater. So long as the back pressure generated by these restrictions do not interfere with the SFC density regulation in the high pressure separation region, a great deal of tunability is possible for optimizing heat transfer. 
     Fourth, due to the expansion, linear velocities of the depressurizing fluid increase dramatically in the transfer tubing. Residual liquids of the system are moved along the flow path largely by shear forces from the expanding gas. This turbulent environment is ideal for the creation of aerosols, whereby very small droplets of modifier liquid are entrained in the gas phase as a “mist”. It is a finding of this study that the aerosol formation within the transfer tubing can be almost completely controlled by proper temperature control of the expanding two-phase system. Aerosol formation is a greater problem at lower temperatures. It is a surprising finding of this work that higher levels of organic modifier with correspondingly lower CO2 content require higher temperature levels to prevent visible aerosol formation. 
     In the preferred exemplary embodiment, the SFC collection system is composed of a moderately restrictive, thermally regulated stainless steel transfer tube which extends from a back pressure regulation component of the SFC chromatograph into a multi-port distribution valve and from the valve to a variety of flowpaths leading either through discrete collection chambers or directly connected to a vented common waste container. 
     Initial separation of the liquid phase sample from carbon dioxide gas occurs immediately at the point of initial decompression within the backpressure regulator of the SFC or SFE instrument. By providing downstream restriction, a minimum backpressure sufficient to prevent the formation of solid CO2 can be maintained while liquid CO2 is present in the transfer lines. 
     The remainder of the CO2 evaporation and separation from the organic modifier occurs in the stainless steel transfer tubing prior to entering the cassette. This is accomplished by exposing the transfer tubing to a series of one or more heaters designed to optimize thermal transfer to the fluid. Ideally, this heater series transfers sufficient energy to the liquid CO2 portion of the emerging fluid to allow for complete evaporation of the liquid CO2 and raise the fluid temperature sufficiently to prevent the transfer tubing from icing externally. Because rates of heat transfer are time dependent, it is beneficial to slow the velocity of fluids within the heater series. 
     During the CO2 evaporation process within the first heated zone, significant separation between the gaseous CO2 and liquid modifier occurs. However, the separation to pure CO2 and pure organic modifier is never realized for several reasons. First, some organic modifier is typically also evaporated into the gas state. The degree of evaporation is largely dependent on the absolute temperature of the fluids within the transfer tubing. While organic modifier evaporation does lead to lower recovery of liquid phase, it does not necessarily reduce the recovery of dissolved components of interest which do not typically have low enough boiling points to convert to vapor. Second, a fraction of CO2 will remain dissolved in the organic liquid. Both temperature and pressure determine the amount of residual CO2. Higher temperatures reduce CO2 solubility while higher pressures increase CO2 solubility. 
     Aerosol formation of the liquid phase is a common problem in SFC sample collection and is a primary cause of loss of the organic liquid phase that contains the dissolved components of interest. Higher temperatures reduce the aerosol generation. The composition of the separated phases also is a factor. Higher temperatures are required to eliminate aerosols in streams with higher organic liquid composition. An additional heated zone is used to trim the fluid temperature to control aerosols. In addition, this heater provides a fine level of temperature control of the fluid before collection in the pressurized collection chamber. As mentioned above, a secondary effect is that a higher trim temperature can reduce the concentration of dissolved CO2, thereby reducing the possibility of uncontrolled or explosive outgassing of the CO2 when the pressure is removed from the collection chamber. 
     Following the trim heater, a valve system is used to divert the biphasic flow stream sequentially to waste or to one of the collection chambers in a collection cassette. The valve system is comprised of one or more valves and an electronic controller. The system is designed to offer rapid response to a manual or automated start/stop signal. Typically, the signal would result from detection of a component of interest emerging from the high-pressure flow system. A start signal would be generated at the initial detection of the component while a stop signal would be generated at the loss of detection. The effect of a start signal is to divert the flow to the first unused collection chamber of the cassette. The effect of the stop signal is to divert the flow to waste. Another possible type of start/stop signal may be based on a timetable rather than physical detection of components. The controller may also have features to limit the access time or flow volume allowed to an individual chamber. In addition, the controller may allow or prevent the system from cycling back to the original chamber if more fractions are desired than there exists available collection chambers. 
     The collection cassette is a resealable apparatus that contains one or more hollow collection chambers open at the top. In the preferred exemplary embodiment, each chamber holds a removable inert liner. The liner collects a fraction of the original sample dissolved in a liquid solvent base. A preferred exemplary embodiment of a cassette has four chambers housing four test tube vials that function as chamber liners. The number of chambers in a cassette may be varied with no effect on performance. Each test tube vial may hold up to its capacity of a separated sample fraction from the high-pressure flow stream. 
     In the preferred embodiment, sample fractions are collected in one chamber of the cassette at a time. The biphasic fluid enters a chamber via a transfer line from the valve system. The tip of the transfer line is preferentially positioned tangential to the inner wall of the collection tube and with a slight downward angle, usually less than 45 degrees from horizontal. Attached to the transfer line and suspended inside a test tube is a guiding spring wire. The spring wire is bowed away from the transfer line and functions as a guide for the transfer line as it descends into a vial. When transfer tubing is properly inserted into a test tube vial, the bowed section of the spring wire engages the circumferential edge of the open end of a test tube vial. As the tubing continues into the test tube, the spring wire compresses against the inner surface of the test tube vial and pushes the tubing towards the opposite side of the vial. As a result, the angled tip of the transfer tubing is pressed against the inner wall of the test tube vial. 
     Both the organic liquid and CO2 gas follow a descending spiral path along the inner wall to the bottom of the collection liner. The liquid collects at this point and begins to fill the liner. The CO2 gas continues in a path up the center of the liner to a vent in the collection chamber. A restrictive transfer line attached to the vent causes the CO2 gas to pressurize the collection chamber both inside and outside the collection liner. The degree of back pressurization within the chamber is roughly proportional to the composition of CO2 in the original mobile phase. 
     The pressurization of the collection chamber serves to slow down the velocity of the CO2 entering the chamber. This in turn reduces the magnitude of shear forces occurring between the CO2 gas and the collected liquid at the bottom of the liner. With lower shear forces, there is less tendency for the collected liquid to become an aerosol and to be removed from the collection tube with the exiting gas. A similar effect is obtained by the proper angling the inlet transfer line relative to the collection tube wall. The closer the angle of the tube is to horizontal the lower the observed turbulence at the liquid surface. However, enough angle must be provided to insure the majority of effluent is directed downward rather than upward on the liner wall. The two effects of back pressure and delivery angle combine to reduce aerosol formation in the collected liquid fraction. The success of optimizing these effects determines how close the inlet tube can come to the collection liquid, and thereby determining how high the liner may be filled before sample loss becomes a problem. When flow to the chamber is stopped, the chamber depressurizes. Once the sample chamber is depressurized, the liner may be removed by opening the top lid of the cassette. 
     The collection of fractions into disposable liners of collection chambers may be automated through the use of robotics. An automated system enables rapid substitution of test tube vials into and out of collection chambers and long unattended run times based on a quantity of vials available for substitution. A programmable robot automatically sequences cassettes between sample injections, thereby speeding up the process while reducing the margin for error. The automated system can collect on the order of thousands of fractions per month. 
     The automated system is contained in laboratory grade housing. The system is comprised of a robotic arm, a supply of test tube vials arranged upright in racks, and an automated version of a cassette assembly. In addition, the system may contain sufficient probes, valves and sample containers to achieve automated delivery of unfractionated samples into the chromatographic or extraction system. 
     The collection cassette and its automated mechanisms are designed for rapid sample collection and minimal stop time between chamber liner replacements. The cassette in the preferred embodiment has two banks of four collection chambers each. A lid is positioned above one bank of collection chambers in the cassette. The lid has four partially recessed annular bores corresponding to the four collection chambers in the cassette. The lid raises and lowers with action from pneumatic actuators mounted on the base of the housing and located on opposite longitudinal ends of the lid. As the actuators simultaneously lower the lid onto the collection cassette, the top edge of each chamber engages the bottom edges of the lid corresponding to the rims of each partially recessed bore. The lid and chambers engage and form pressure tight seals in each chamber in preparation for sample fraction collection. The lid has transfer and waste line tubing passing through each recessed bore that correspond to each collection chamber. Each tubing pair enters a test tube as the lid is lowered onto the cassette. The spring wire attached to the inlet tubing guides an inlet tube into a test tube vial. An angled tip on the tube is forced against the inner wall of the test tube. After the lid has sealed on the row of collection chambers, a valve system dispenses the flowstream containing gaseous and liquid phases into the chamber liners from the sample fractionation process. 
     When all test tube vials in the pressurized cassette row have been filled and depressurized, the lid lifts off of the cassette. The cassette then moves laterally, or shuttles, until a row containing empty collection chamber liners is moved under the lid in place of the former row. The cassette is constrained to shuttle laterally along a path on the base of the housing. The lid lowers and engages the new row of chambers, thereby preparing the test tubes to accept sample fractions. Meanwhile, the former row of chamber liner test tube vials containing liquid fractions are removed from the collection chambers and transported to open spaces in a storage tray via a robot arm. 
     In summary, samples in the preferred embodiment are dissolved in a minimum volume of modifier solvent and are collected in removable and reusable liners. Through controlling flowrate, velocity, temperature, and pressure in the system, superior separation of near-supercritical elution fluid is obtained. Collection efficiencies of up to 98% of injected sample components may be realized. The cassette, by utilizing pressurized collection chambers and disposable liners in the process, minimizes the use of additional collection and cleaning solvent spent by a laboratory, which is economical and good for the environment. Laboratories and research facilities that demand purity of samples while maximizing output and minimizing waste will benefit from the proposed invention. Large-scale sample fractionation and collection, numbering in the thousands of samples per month, may be realized from the exemplary embodiment. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     For a better understanding of the nature of the present invention, reference is had to the following figures and detailed description, wherein like elements are accorded like reference numerals, and wherein: 
     FIG. 1 illustrates a schematic flow diagram of the supercritical fluid chromatography system and the collection system including the sample cassette embodied in the invention. 
     FIG. 2 illustrates an exploded isometric view of a sample collection cassette. 
     FIGS. 3A and 3B illustrate top and bottom plan views of the cassette lid. 
     FIG. 4 illustrates a plan view of an alternative exemplary embodiment of an automated fraction collection system. 
     FIG. 5 illustrates a side view of an alternative exemplary embodiment of an automated fraction collection system. 
     FIG. 6 illustrates an exploded isometric view of a shuttle sample collection cassette, lid, and mechanized controlled movement system. 
     FIG. 7 illustrates a detailed side view of the shuttle cassette and associated mechanical control apparatus. 
     FIGS. 8A and 8B illustrate detailed cross sectional views of transfer tubing before and after insertion into a test tube vial. 
     FIG. 9 illustrates an alternative embodiment of an integrated collection cassette having multiple rows of collection chambers. 
     FIG. 10 illustrates an additional alternative embodiment of a shuttle collection cassette for an automated system. 
    
    
     DETAILED DESCRIPTION OF PREFERRED EXEMPLARY EMBODIMENTS 
     The preferred embodiment of the apparatus is illustrated in the flow chart of FIG. 1 within the perimeter line  10 . Except where noted, specifications for a preferred exemplary embodiment are given for a system that accepts flows of 20 to 100 mL/min total flow (CO2 plus modifier flow) in the highly compressed state from the pumping system. Flowrates for alternative embodiments could range in orders of magnitude higher or lower through adjustment or substitution of system hardware and flow parameters. 
     In the preferred exemplary embodiment, the SFC collection system is composed of a moderately restrictive, thermally regulated transfer tube  12  which extends from a back pressure regulator  14  into a multi-port distribution valve  22  and from the valve to a variety of flowpaths leading either through discrete collection chambers  32  or directly connected to a vented common waste container  26 . 
     Expanded elution fluid leaves the backpressure regulator  14  at a velocity of approximately two to five times the flow velocity upstream of the backpressure regulator  14  and under back pressure of approximately twenty to forty bars. Variations in the expansion occur as a result of the changing modifier solvent concentration from 2.5 to 50 percent over the course of a separation. 
     Initial separation of the liquid phase sample from carbon dioxide gas occurs immediately at the point of initial decompression within the backpressure regulator  14  of the SFC or SFE system. By providing downstream restriction, a minimum backpressure sufficient to prevent the formation of solid CO2 can be maintained while liquid CO2 is present in the transfer lines  12 . The degree of CO2 evaporation is a function of both the available heat transfer in this region and the downstream flow restriction which limits the amount of expansion available to the decompressing fluid. Due to the pressure drop across the backpressure regulator  14 , a fraction of the emerging CO2 will evaporate, typically causing a significant drop in the temperature of the emerging fluid. 
     Further separation and evaporation of CO2 from the organic modifier occurs in stainless steel transfer tubing  12  running between the first backpressure regulator  14  and the cassette  24 . The transfer tubing  12  containing a flowstream of the biphasic CO2 and modifier is exposed to a series of a heaters  16 ,  18  designed to optimize thermal transfer to the biphasic fluid in the flowstream. Ideally, this heater series transfers sufficient energy to the liquid CO2 portion of the emerging fluid to allow for complete evaporation of the liquid CO2 and raises the fluid temperature sufficiently to prevent ice from forming externally on the transfer tubing  12 . 
     During the CO2 evaporation process within the first heated zone, significant separation between the gaseous CO2 and liquid modifier occurs. However, the separation to pure CO2 and pure organic modifier is never realized. Some organic modifier is typically evaporated into the gas state. The degree of evaporation is largely dependent on the absolute temperature of the fluids within the transfer tubing  12 . While organic modifier evaporation does lead to lower recovery of liquid phase when it reaches the collection cassette  24 , it does not necessarily reduce the recovery of dissolved components of interest which do not typically have low enough boiling points to convert to vapor. A fraction of CO2 will also remain dissolved in the organic liquid modifier. Both temperature and pressure determine the amount of residual CO2. Higher temperatures reduce CO2 solubility while higher pressures increase CO2 solubility. Turbulent flow of the CO2 gas within the narrow tubing also produces a strong shearing force that propels the liquid down the walls of the transfer tube  12 . This very turbulent flow frequently causes small droplets at the liquid surface to rip away from the bulk liquid and become entrained into the rapidly moving gas phase of the fluid down the transfer tube  12 . Such an effect is called aerosol formation, or “misting”. 
     A plurality of heaters may be mounted in series to heat the elution fluid. In FIG. 1, the preferred exemplary embodiment has an evaporator heater  18  and a trim heater  20  mounted in series after the backpressure regulator  14 . The evaporator  18  is heated with an appropriately sized cartridge heater and controlled by an appropriate heater controller. In the preferred embodiment, transfer tubing  12  is tightly coiled around the heating assembly and optimized for thermal contact. The elution fluid is heated to within the control temperature of the evaporator  18 , which is between approximately 5 to 50 degrees C., to protect heat sensitive compounds from being damaged. The objective is to boil CO2 out of the elution fluid as the fluid passes through the evaporator  18 . To complete the required heat transfer, biphasic elution fluid inside transfer tubing  12  enters the final heat exchanger, which is a trim heater  20 . In the preferred embodiment, the trim heater setting is typically above the evaporator  18  setpoint. The heater  20  is used not only to suppress aerosol formation within the transfer tube  12  but also to control the level of dissolved CO2 in the liquid phase. 
     It is beneficial to slow the velocity of fluids within the transfer tubing  12  passing through the heater series  18 ,  20 . The fluid velocity is slowed inside the transfer tubing  12  by placing a restrictive orifice or smaller diameter tube immediately downstream from first heater series. Elution fluid exits the evaporator  18  and enters a flow restrictor  16 , which provides a higher backpressure in the evaporator  18  and thereby slows the flow and increases the contact time of the liquid CO2 phase. The restrictor  16  also insures a high enough backpressure to prevent the liquid carbon dioxide from forming solid carbon dioxide, also known as dry ice. The restriction increases the backpressure in the heated zone and reduces the amount of the gas expansion. In an alternative exemplary embodiment, the velocity of fluids can be slowed after all heaters, however such a configuration does not control the final expansion of CO2 which can result in uncontrolled cooling of fluids within the transfer lines. As a result, the ability to actively suppress aerosol formation may be diminished. 
     After exiting the trim heater  20 , transfer tubing  12  connects to the common port of a valve system  22 . The valve system in the preferred exemplary embodiment is a multi-port selector valve  22 . As elution fluid from the peak of interest passes through the valve system  22 , the gas and liquid phases are directed into either a collection cassette  24  or to a waste stream container  26 . The outlet ports on a multi-port selection valve  22  are connected to a plurality of transfer tubing lines  28 . The transfer lines  28  pass through a cassette lid  30  and into discreet chambers  32  within the cassette  24 . The transfer lines  28  have airtight and pressure resistant connections into and out of the cassette lid  30 . The remaining ports in a multi-port selection valve  22  connect to waste transfer lines  34 . In an alternative exemplary embodiment, multiple discreet valves are installed and connected to the incoming transfer line  12 , having each valve port connected to an individual collection chamber  32  in the cassette  24  and a discreet valve connected to a waste line  34 . 
     Inlet lines  28  entering a collection chamber  32  insert into a test tube vial  36  within a chamber  32 . Liquid phase  38  is captured in a test tube  36  while gaseous phase escapes out of a chamber  32  through a discharge line  40 . Gas in the discharge line  40  is flowing at high pressure. Discharge lines  40  from the cassette  24  run through a pressure relief switch  42  to protect the cassette and upstream components from possible damage due to over-pressurization from a system malfunction. 
     Referring additionally to FIG. 2, a preferred exemplary embodiment of the cassette  24  comprises four discreet collection chambers  32 . However, in alternative embodiments, one or more individual chambers  32  are possible in the cassette  24 . Each collection chamber  32  in the preferred embodiment is a closed system that is the final separation point of liquid and gaseous phases. Chambers  32  are hollow cylinders constructed of high strength transparent plastic to allow visual monitoring of separation and collection processes. The cassette chambers  32  can be formed of stainless steel or other appropriate laboratory-grade materials. The chambers  32  sit parallel and upright in the cassette  24 . Each chamber  32  is constrained at its upper and lower ends within a molded frame  44 ,  46 . Each chamber  32  is set with the open end surrounded by the upper molded frame  44  and the lower end partially embedded into the lower molded frame  46 . Communication of liquid or gaseous phases between chambers  32  is prohibited by seals  48  that are seated in a groove  50  at the top, open end of each chamber  32 . 
     Each collection chamber  32  houses a removable, replaceable liner. A standard glass test tube vial  36  functions as a liner and is seated upright inside each the chamber  32 . The closed bottom of a test tube vial  36  rests on the base of the chamber  32  and is easily removable. Once inserted, the top of the test tube vial  36  must be lower than the combined height of a chamber  32  and the internal recessed bore  60  (FIG. 3) of a lid piece  30  when the lid and cassette  24  are engaged. A test tube vial  36  and a chamber  32  are a single pressurized system that communicate through the top of the chamber  32 . The test tube vial  36  functions as a disposable liner for the chamber  32  to capture the liquid phase  38  that has separated from the flow stream. The inside of the vial  36  and the annular space of the chamber  32  surrounding the vial are equilibrated to the same pressure, which is a range of approximately 20 to 100 psig during separation processes for a flowstream up to 50 ml/min. This arrangement enables sample fraction collection at high pressure using standard laboratory glass test tube vials  36  without a risk of breaking the glass vial inside the chamber  32 . 
     FIG. 2 illustrates the cassette  24 , comprising a rectangular frame securing four upright chambers  32 . The upper section  44  and lower section  46  of the molded frame hold the chambers  32  in place. The frame is completed by two rigid rectangular end pieces  52  attached to the upper and lower sections. Each end piece  52  is a metal plate fastened to the upper  44  and lower  46  frame sections with machine screws  54 . Butterfly latches  56  are installed at the top of both rigid end pieces  52  secure the lid piece  30  to the top of the cassette  26 . The lid  30  may be removed manually between sample injections for quick access to, and removal of, chamber liners  36 . As illustrated in FIG. 1, the bottom of each chamber  32  has a transfer tube or orifice  33  running completely through the base of a collection chamber and lower frame  46 . The orifice  33  through the chamber base  46  can be used to remove liquid phase fluid from a chamber  32  without opening the chamber or depressurizing the chamber  32 . The sample discharge port  33  also permits easier draining and cleaning of the chamber  32  during maintenance of the cassette  24 . 
     FIGS. 3A and 3B illustrate top and bottom views of the removable cassette lid  30 , respectively. The lid  30  has four sets of three boreholes  58  in a triangulated pattern positioned such that each set of boreholes is directly over each of the chambers  32  when the lid  30  is engaged to the cassette  24 . The bottom face of the lid  30  has partially recessed bores  60  positioned directly above each chamber  32  when the lid  30  and cassette  24  are engaged. The diameter of a recessed bore  60  is sized slightly smaller than chamber  32  diameters. The recessed bore&#39;s  60  perimeter is positioned completely inside of a seal  48  when the lid  30  is fastened to the cassette, as illustrated in FIG.  2 . The recessed boreholes  60  allow a test tube  36  to stand taller than the top planar surface of the upper frame section  44  of the cassette  24  so that a test tube  36  may be removed without reaching into a collection chamber  32 , thereby possibly cross-contaminating subsequent samples. To guide the lid  30  and cassette base  24  together when engaging, alignment pins  62 , illustrated in FIG. 3, are formed on the outer, top surface of the cassette frame  24 . Partially recessed bores  63  in the lid  30  receive the alignment pins  62  from the cassette frame  24 . Catches  64  for the butterfly latches  56  are attached to each long end of the lid  30 . 
     Inlet transfer tubing  28  carries liquid and gaseous phases into test tube vials  36  housed in each collection chamber  32  of the cassette. Each inlet tube  28  fits through a hole  58  in the lid  30  and inserts into a test tube vial  36 . Proper fittings on the tubing  28  provide airtight connections that can also withstand pressure forces in the SFC system. Inlet tubing probes  66  direct elution fluid into a test tube vial  36  and an outlet tube  68  provides an escape route for gas that is under pressure to exit the chamber  32  and discharge to waste collection  26 . 
     In the preferred embodiment, fractions are collected in one chamber  32  of the cassette  24  at a time. During the fractionation process, both the liquid phase and the gas phase discharge into the collection vial  36  where final separation takes place. The pressurization of the collection chamber  32  serves to slow down the velocity the CO2 within the chamber  32 . This in turn reduces the magnitude of shear forces occurring between the CO2 gas and the collected liquid at the bottom of the liner  36 . With lower shear forces, there is less tendency for the collected liquid to become an aerosol and removed from the collection liner  36  with the exiting gas. A similar effect is obtained by the proper angling the inlet transfer line relative to the collection liner  36  wall. The closer the angle of the tube  66  is to horizontal the lower the observed turbulence at the liquid surface. However, enough angle must be provided to insure the majority of effluent is directed downward rather than upward on the liner  36  wall. 
     The biphasic elution fluid enters a chamber  32  via a transfer line  28  from the valve system  22 . As illustrated in FIGS. 8A and 8B, the tip of the transfer tube  66  is a probe preferentially positioned tangential to the inner wall of the collection vial  36  and with a slight downward angle, usually less than 45 degrees from horizontal. Attached to the probe  66  is a guiding spring wire  70 . The spring wire  70  is bowed away from the probe  66 . The spring wire  70  acts as a guide for the probe  66  as the probe descends into a test tube vial  36 . When the probe  66  is properly inserted into a test tube vial  36 , the bowed section of the spring wire  70  contacts the circumferential edge of the open end of a test tube vial  36 . As the tubing  66  continues into the test tube vial  36 , the spring wire  70  compresses against the inner surface of the vial  36  and pushes the probe  66  towards the opposite side of the vial  36 . As a result, the angled tip of the probe  66  is pressed against the inner wall of the test tube vial  36 . 
     The spring wire  70  is extruded from inert materials that will not chemically interfere with collected samples in the test tube vials  36 . In an alternative exemplary embodiment, the probe section  66  of the transfer tubing  28  is a rigidly held stainless steel probe attached to the cassette lid  30 . Metal versions of probe  66  may be terminated with a larger OD Teflon tube sleeved onto the metal probe to prevent scratching and possible rupture of the inner wall of the collection liner  36 . 
     Both the organic liquid and CO2 gas follow a descending spiral path along the inner wall to the bottom of the collection liner  36 . The liquid phase collects at this point and begins to fill the test tube vial  36 . The CO2 gas continues in a path up the center of the vial  36  to a vent through the top of the collection chamber  32 . A restrictive transfer line attached  72  to the vent causes the CO2 gas to pressurize the collection chamber  32  both inside and surrounding the collection liner  36 . The degree of back pressurization within the chamber is roughly proportional to the composition of CO2 in the original mobile phase. 
     The two effects of back pressure and delivery angle combine to reduce aerosol formation in the collected liquid fraction. The success of optimizing these effects determines how close the inlet tube  66  can come to the collection liquid, and thereby determining how high the liners  36  may fill before sample loss becomes a problem. When flow to the chamber  32  is stopped, the chamber depressurizes. Once a chamber  32  is de-pressurized, the test tube vial  36  containing liquid phase may be removed by opening the top lid  30  of the cassette  24 . 
     The outlet line tubing  72  from each chamber  32  is connected to a fixed restrictor  42  to keep pressure inside the chambers  32 . The fixed restrictor  42  raises the upstream pressure between approximately 20 and 100 psig depending on CO2 flow rate. Each discharge line  72  passes through a pressure switch  78  to protect against overpressuring and rupturing. Pressure in each chamber is monitored visually with a pressure gauge  76  that is threaded into the lid  58  over each chamber  32 . Discharge lines  72  are directed to a waste collection tank  26 , from which the CO2 is vented. To increase laboratory safety, the system should not have any exposure of waste effluent, samples, or vented CO2 to ambient laboratory air. The liquids and gasses in the system remain in a contained system that can be directed to a hood or safety exhaust  26  to maximize safety for the technician. 
     The volume of the captured fractionated liquid phase  38  in the collection vial  36  is controlled manually or automatically. Automatic control in the preferred exemplary embodiment of the valve system  22  and is comprised of one or more valves and an electronic controller. The valve system  22  is designed to offer rapid response to a manual or automated start/stop signal. A signal can result from detection of a detection of a component of interest emerging from the high pressure flow system. A start signal would be generated at the initial detection of the component while a stop signal would be generated at the loss of detection. The effect of the stop signal is to divert the flow to waste lines  26  or to another chamber  32 . An alternative embodiment of a type of start/stop signal may be based on a time-table rather than physical detection of components. The controller may also have features to limit the access time or flow volume allowed to an individual chamber  32 . In addition, the controller may allow or prevent the system from cycling back to the original chamber  32  if more fractions are desired than there exist available collection chambers  32 . 
     An alternative exemplary embodiment of the collection cassette and system is illustrated in FIGS. 4 through 7. This embodiment is an automated system that utilizes a robotic arm  80  to replace chamber collection liners  36  after filling with sample fractions. The robotically controlled unit is designed for rapid filling and replacement of chamber liners  36  combined with a long unattended run time. Supply trays  86  of clean test tube vials  36  that function as chamber liners  36  are located within the unit&#39;s housing  82 . A robotic arm  80  is controlled to replace one or more liners  36  from a row of collection chambers  32  in a collection cassette  84  with liners  36  from a fresh supply rack  86 . The robotic arm  80  is mechanized to replace liners  36  on a first row of the cassette  84  while liners  36  on a second row are automatically moved into place. This robotically automated alternative embodiment provides faster sample collection through a minimum of down time to replace liners  36  as well as the ability to collect a greater number of samples during an unattended session. 
     FIGS. 4 and 5 illustrate the plan and side views, respectively, of an automated alternative exemplary embodiment of the SFC sample collection system. The components for the system are partially enclosed with a laboratory-grade housing structure  82  having a raised mounting base  88  within the housing  82 . The housing  82  is supported with adjustable feet  90  that are distributed around the base of the housing  82 . The feet  90  adjust the level the housing  82  to compensate for uneven or slanted surfaces. Supplies of uncontaminated test tube vials  36  are stored in racks  86  placed on a raised interior base  88  of the housing  82 . Each test tube vial  36  is held upright and secured in-place in a rack  86  by molded supports. Each support rack  86  consists of circular sections attached tangentially to neighboring sections, forming multiple rows and columns. The molded supports loosely secure test tube vials  36  that are held in each circular opening of the racks  86 . The vials  36  are maintained equidistant from each neighboring vial to provide adequate spacing for a grabbing jaw  92  on a robotic arm  80  to grasp a vial  36  without interference from a neighboring vial. The spacing also prevents chipping or breakage during movement and replacement of the rack  86 . Two racks  86  of test tube vials  36  are illustrated in the Figures, however the system could easily expand to a plurality of racks of the vials  36 . 
     An alternative exemplary embodiment of a cassette  84  and associated system devices is installed on the raised interior base  88 . The cassette  84  has a plurality of rows of chambers that are constrained to lateral movements that are automatically controlled with a pneumatic actuator  96 . This cassette  84  is termed the “shuttle cassette”, or simply “the shuttle.” FIGS. 6 and 7 illustrate the shuttle cassette  84  in isometric and side views, respectively. The shuttle cassette  84  is constructed similar to the exemplary embodiment with an added row of collection chambers  102 . The shuttle  84  comprises upper and lower rectangular molded frames  98 ,  100  supporting a plurality of rows of upright cylindrical collection chambers  102 . The shuttle  84  is constructed with two rows of four cylindrical collection chambers  102  in each row. The size of the shuttle  82  can be modified to add additional rows of chambers  102  or additional chambers per row, such as an alternative embodiment featuring three rows of chambers  102  illustrated in FIG.  10 . The shuttle cassette  84  is formed on two opposite ends with rigid rectangular plates  104 . Each end plate  104  is fastened to the upper  98  and lower  100  molded frame sections with machine screws  106 . The shuttle  84  may be constructed with permanent attachments and fittings, however, a shuttle that readily disassembles allows easier and thorough cleaning and replacement of worn or damaged components. 
     The collection chambers  102  are formed of high-strength transparent plastic, which allows visual monitoring of the collection process inside of each chamber  102 . As an alternative, the chambers  102  may be formed of stainless steel or a similar high-strength material compatible with SFC parameters described herein. Each cylindrical chamber  102  is set into the lower molded frame  100  for base support. The upper molded frame section  98  is secured near the open, top end of each chamber  102 . Each chamber  102  extends above the top surface of the shuttle  84  at a standardized distance adequate to seal the chambers  102  with an automated lid piece  108 . Standard laboratory test tube vials  36  may be inserted into each of the chambers  102  to act as a removable or disposable liner for each chamber. 
     The automated shuttle cassette  84  is constrained to lateral movements on the inner raised base  88 . The lower molded frame section  100 , or base, of the shuttle cassette  84  has an horizontally bored hole  110 , illustrated in FIG. 7, running perpendicular to the open sides of the shuttle. Offset from the shuttle  84  is an actuator  96  installed on the raised base  88  of the housing unit  82 . Attached to the actuator  96  is rod  94  or controller arm. The rod  94  is constructed of a rigid material, such as stainless steel, and inserts into the bored hole  110  in the base of the shuttle cassette  84 , wherein it is firmly attached to the base frame  100 . The actuator  96  executes lateral movements of the shuttle  84  according to commands sent from a programmable control system. In an alternative embodiment, the base of the shuttle  100  has small rollers  112  installed around the base, as illustrated on FIG.  7 . The rollers  112  are guided laterally by grooved tracks in the base of the housing  88 . The tracks not only constrain the movement of the shuttle  84  but also remove tension from the controller arm  94  and actuator  96  gears caused by the shuttle  84  drifting into angled movements caused by uneven friction on the rollers, initial off-center displacement after shuttle  84  installation, or irregularities on the surface of the housing base  88 . Other methods of providing constrained lateral movement are possible in alternative embodiments, such as utilizing guide tracks wherein guides on the shuttle  84  are enclosed within tracks riding on ball-bearings. 
     Referring to FIGS. 6 and 7, the lid  108  of the shuttle cassette  84  is automatically controlled to engage a row of collection chambers  102  after the shuttle is moved into place directly below the lid  108  by the lateral actuator  96 . In the alternative embodiment, the lid  108  is constructed of stainless steel. However, high density plastic, or a similar material having equivalent rigidity and composition for use in the collection system, is sufficient. The lid  108  has a hole  114  through each longitudinal end, bored parallel to the vertical axis of the lid. The holes  114  in each end of the lid  108  are sized to fit a threaded rod  116 . Two nuts  118  threaded above and below the lid  108  secure the lid to each rod  116 . The lid  108  is constrained to move only in the vertical plane. The movements of each rod  116  are controlled by actuators  120  mounted to the raised base of the housing  88 . The two pneumatic actuators  120  controlling the lid movements are synchronized to move the rods  116  vertically, thereby raising and lowering the lid  108  onto a row of collection chambers  102  in the shuttle cassette  84 . 
     FIG. 7 illustrates the lid piece  108  raised above the shuttle  84  prior to engagement. The bottom face of the lid  108  has four bores  122  partially recessed into the lid corresponding to four chambers  102  in a row of the shuttle. As the lid  108  is lowered by the pneumatic actuators onto the shuttle  84 , each chamber  102  of a row partially inserts into a recessed borehole  122 . The lid  108  stops at a programmed point at which the circular edge of each bore  122  engages and seals against the flat upper surface of the shuttle frame  98 . Each partially recessed borehole  122  in the lid  108  has a diameter larger than the chamber&#39;s  102  diameter. As the lid  108  lowers onto the shuttle  84 , the recessed boreholes  122  are lined up with the top, open ends of the chambers  102 . The larger diameter recessed boreholes  122  each totally enclose the open end of each chamber  102 . An appropriate sealing  0 -ring or similar component is placed around the top of each chamber  102 , between the top of the shuttle  84  and the lid  108 , to provide an airtight and pressure resistant seal when the two components engage. Alignment pins  124  are located on the top surface  98  of a shuttle  84  at both ends of each row of chambers  102 . The pins  124  are shaped as half-spheres on the top surface of the shuttle  84  and provide additional protection for shuttle collection chambers  102  from misalignment of the shuttle  84  to the lid  108 . As the lid  108  engages onto the shuttle  84 , the alignment pins engage corresponding bores  126  in the lid. 
     A collection chamber  102  is a discreet system that is the final separation point of liquid and gaseous phases. Communication of liquid or gaseous phases between chambers  102  is prohibited through the lid  108  that seals each chamber airtight as it automatically lowers onto a row of chambers in the shuttle cassette  84 . Similar to the exemplary embodiment of the cassette, each chamber  102  in the shuttle  84  holds a chamber liner  36  to catch fractionated liquid phase. The liner  36  is a standard laboratory test tube vial  36 . The closed bottom of the test tube  36  rests at the base of each chamber  102 , which rests on the lower molded frame of a shuttle  100 . A test tube vial  36  and chamber  102  communicate as a single pressurized system. FIG. 8B illustrates the position of the open end of a vertically disposed test tube vial  36  below the top of a recessed borehole  122  after the lid  108  engages the shuttle  84 . The inner pressure of the test tube vial  36  and the chamber&#39;s  102  annular space surrounding the vial are equilibrated and range from approximately 20 to 100 psig during collection processes. This arrangement enables sample fraction collection at high pressure using standard lower pressure glass or plastic vials by equilibrating the pressure forces inside and outside the vial  36 . 
     As illustrated in FIG. 7, the lower, closed end of each chamber  102  has a sample discharge port  128  running completely through the lower shuttle frame  100 . A plug is inserted into each sample discharge port  128  during regular use of the shuttle  84 . The sample discharge port  128  permits removal of liquid phase that is collected directly into a chamber  102  without using a liner. By withdrawing liquid phase through the sample discharge port  128 , the liquid phase may be collected without disengaging the lid  104  from the shuttle  84 . Liquid phase may be evacuated from a chamber  102  under pressure or gravity fed out of a chamber after chamber depressurization. 
     Inlet  66  and outlet  68  tubing for transferring influent and effluent liquid and gas phases between the shuttle cassette  84  and external transfer lines are illustrated in FIGS. 6 and 7. Inlet  66  and outlet  68  tubing for the shuttle  84  pass through the lid  108 . Transfer tubing  66 ,  68  is constructed from high-pressure stainless steel or equivalent materials. Inlet tubes  66  carry gaseous and liquid phases into a collection chamber  102  under high pressure. Outlet tubes  68  carry separated gaseous phase to a waste tank  26  for venting or disposal. The lid section  108  has four sets of three holes  134  in triangular formations that pass through the lid and are located to correspond with collection chambers  102  when the lid is engaged to the shuttle cassette  84 . 
     In addition to transfer tubing, one of the holes  134  permits measurement of pressure forces inside a chamber with a pressure gauge  76  threaded into the hole  134  from top of the lid  108 . The transfer tubing  66 ,  68  and pressure gauge  136  all have pressure resistant airtight fittings specified to withstand pressure forces created in the SFC system. Transfer tubes  66 ,  68  installed below the lid  108  insert into a test tube vial  36  when the lid  108  is engaged to the shuttle cassette  84 . The tip of each inlet tube  66 , or probe, is constrained to an angle less than 45 degrees and wrapped with non-reactive spring wire  70  that is bowed along the vertical section, similar in construction and purpose as described in the preferred embodiment. The spring wire  70  serves to angle the inlet tubing  66  inside a test tube vial  36  by applying pressure forces against the vial&#39;s  36  inner wall. As a result, the open tip of the inlet tube  66  is forced tangentially against an opposing inner wall of the vial  36 . This configuration of the inlet tube  66  is desirable because it causes the liquid phase that exits the inlet tube  66  to contact a side wall of the vial  36  and swirl down the inner wall of the vial  36  in a spiraling motion. The swirling action provides the final separation process of liquid phase from entrained gaseous phase while preventing re-entrainment and loss of sample fractions from the liquid phases into gaseous phases or aerosol mists that can be carried away with gaseous phases to a waste vent  26 . 
     In an alternative exemplary embodiment, a robotic arm, such as a Cartesian or three-dimensional robotic arm, is programmably controlled to move test tube vials between supply racks and the shuttle cassette collection chambers. FIGS. 4 and 5 illustrate a three-dimensional robotic arm  80  mounted to a wall of the unit housing  82  near the shuttle cassette  84 . A host PC or microcontroller issues positioning commands for the arm&#39;s movement and controls automated functions. The arm  80  has a jaw  92  to grab and place test tube vials  36  into the shuttle cassette  84  from the test tube supply racks  86 . The jaw  92  is controlled to grip test tube vials  36  of specific outer diameter and at specific locations within the unit  82 . In the alternative embodiment illustrated in FIG. 5, the robotic arm  80  is gripping one test tube  36  in its jaw  92  to move the test tube between the shuttle  84  and a supply rack  86 . To increase the volume of vials  36  exchanged, the gripper jaw  92  could be modified to grip two or more test tube vials, multiple jaws could be placed on a single arm  80 , or multiple robotic arms could work on the same embodiment. The arm  80  acts in concert with the automated movements of the shuttle  84 . As a row of chambers  102  in the shuttle  84  is engaged to the lid  108 , the robotic arm  80  replaces test tube vials  36  in the shuttle that are filled with collected sample fractions with fresh vials  36  from a supply rack  86 . When a row of test tubes  36  in the shuttle  84  have been replaced, and the row of vials  36  under the lid  108  have captured liquid phase fractions, a programmable controller signals the pneumatic actuators controlling the lid  120  to disengage and move the lid  108  away from the shuttle  84 . The lateral control  96  of the shuttle  84  is then signaled to move the shuttle such that the row of chambers  102  containing clean, uncontaminated test tube vials  36  correspond to a position underneath the lid  108  prior to engagement. The lid actuators  120  are then signaled to engage the lid  108  again to the shuttle  84 , thereby preparing the chambers to receive liquid phase fractions. The robotic arm  80  next grabs vials  36  from the exposed shuttle chambers  102  that contain liquid phase fractions and places them into a supply rack  86 . The arm  80  then replaces an uncontaminated vial  36  into each empty chamber  102  until a row of chambers is completely filled with fresh test tubes. This process is repeated for the length of a sample run or until the system is depleted of uncontaminated test tube vials from the supply racks  86 . 
     An alternative embodiment of a collection cassette is illustrated in FIG.  9 . An integrated cassette  140  consists of multiple rows of wells  144  in a grid pattern formed similar to a titration tray. The smaller footprint of the integrated cassette  140  can increase the density of collection chambers over the shuttle cassette  84 . The integrated cassette  140  also functions as a storage tray for gathered liquid phase fractions. Therefore, time and expense are saved during sampling procedures by removing the steps of the substituting chamber liners  36  and replacing liners from a separate storage area. By modifying the lid  108  and mechanics of the automated collection system, the integrated cassette  140  may serve as its own sample collection cassette and storage tray and can rapidly receive fractions without having to replace liners  36  between each sample injection. The robotic arm  80  in the system may replace integrated cassette  140  units as a whole after a sampling event is completed or chamber wells  144  contain the desired amount of liquid phase fractions. A plurality of integrated cassettes  140  are stored in the automated collection system providing the means for hundreds of collected fractions during an automated run. A preferred construction of an integrated cassette is a 4×6 chamber array in the deep-well micro titer plate format used commonly in the pharmaceutical industry. Such a format improves automation storage density not only due to more chambers per area, but these chambers are also easily stackable, which gives an added dimension of sample storage capacity. This alternative embodiment is a shuttle cassette tray  140  formed from high-strength materials such as plastic, resin, or stainless steel. 
     The integrated cassette tray  140  is also advantageous for rapid fraction collection because it can be modified to contain replaceable liners  36  in the wells  144  or use no liners, thereby collection liquid fractions directly into the wells  144 . The integrated cassette  140  can be replaced as a unit after wells  144  are filled with liquid phase fractions. 
     An alternative embodiment of an automated system using a cassette tray would appear similar to that illustrated in FIG. 4 but with certain modifications. Modifications to the automated system include spacing for a supply of cassette trays  40  instead of test tube racks  86 , sizing of the lid piece  108  and associated mechanized controllers  120  and transfer tubing  66 ,  68 , sizing of lateral mechanized controllers  96  for the tray  140  while switching between rows of chambers  144  during fraction collections, and modification of a robotic arm  80  to substitute filled cassette trays  140  with new trays from a supply area. An alternative to this configuration is having a moveable lid section  108  connected to a robotic arm  80  that engages each row of chambers in a supply rack of trays  140  without ever moving the trays. 
     As can be understood from the above description, the sample collection system has several advantages, for example: it provides simplified prep-SFC sample collection; it collects only fractions of interest from the injected sample; it collects purified samples into removable, inexpensive, and disposable collection vials; it provides extremely efficient and controllable gas and liquid phase separation, thereby providing up to 98% consistent sample recovery; it is environmentally friendly and economical because it eliminates additional use of solvents to collect, trap, or recover samples, and clean unnecessary associated mechanical separation equipment; it allows high speed, high volume, and high purity SFC sample collection. 
     Because many varying and different embodiments may be made within the scope of the inventive concept herein taught, and because many modifications may be made in the embodiments herein detailed in accordance with the descriptive requirements of the law, it is to be understood that the details herein are to be interpreted as illustrative and not in a limiting sense.