WO2013062635A2

WO2013062635A2 - Automated conversion between sfc and hplc

Info

Publication number: WO2013062635A2
Application number: PCT/US2012/042844
Authority: WO
Inventors: Edwin E. Wikfors; Kimber Fogelman; Terry A. Berger
Original assignee: Agilent Technologies, Inc.
Priority date: 2011-06-14
Filing date: 2012-06-16
Publication date: 2013-05-02
Also published as: WO2013062635A3

Abstract

An apparatus, system, and process of converting a standard, high performance liquid chromatography (HPLC) flow path to a flow path suitable for supercritical fluid chromatography (SFC) are described. This reversible technique is applied to a variety of flow configurations including binary, high pressure solvent mixing systems and quaternary, low pressure solvent mixing systems than can be conventionally operated or automated. The technique is generally applied to the fields of supercritical fluid chromatography and high pressure liquid chromatography, but users skilled in the art will find utility for any flow system where pressurization components must be periodically applied to and removed from both ends of a flow stream in an automated manner.

Description

AUTOMATED CONVERSION BETWEEN SFC AND HPLC

Inventors: Edwin E. Wikfors, Kimber D. Fogelman, and Terry A. Berger

Background

[0001] Liquid chromatography (LC) is a known separation technique for isolating and identifying individual dissolved components contained typically in a liquid sample. The technique uses a liquid mobile phase flowing past an adsorbent stationary phase to achieve separation. The terms high pressure-, medium performance-, high

performance-, ultra performance- and ultra-high performance liquid chromatography are generally accepted terms related to the pressure ranges and speed of separation achieved by different LC instrumentation. For the purposes of this application, the term high performance liquid chromatography (HPLC) will be used generically to include all forms of liquid chromatography using positive displacement pumps to propel fluids which are liquids at typical laboratory temperatures and pressures, regardless of the maximum pressure ranges these pumps typically achieve. In other words,

abbreviations such as MPLC, UPLC, UHPLC and HPLC common in the art of LC will all be considered under the generic term HPLC. Forms of liquid chromatography not falling under this term would be those where the flow of the liquid mobile phase is driven by gravity, capillary action, pneumatic pressures, centrifugal forces and other means than positive displacement pumps, or where the pumped fluid is not liquid at laboratory conditions of temperature and pressure.

[0002] The most common form of HPLC is reversed phase HPLC or rHPLC which uses mobile phases consisting of water and organic solvents to elute mixtures of compounds through a nonpolar stationary phase, such a C18 with the least polar compounds eluting last. These more highly retained compounds in rHPLC can be eluted faster by reducing the polarity of the mobile phase. A less common form of high performance chromatography is supercritical fluid chromatography (SFC), which operates in the same typical pressure ranges as HPLC but instead uses pressurized carbon dioxide at liquid-like densities combined with organic solvents to perform normal phase separations. As the names suggest, normal and reversed phase separations have opposing mechanisms where normal phase uses polar stationary phases and increasing polarity of mobile phase to encourage elution of later eluting polar

compounds. Packed column SFC (pSFC) using typical normal phase and chiral phase HPLC columns is the most accepted form of SFC in use today. Because of the different mechanisms, the rHPLC and pSFC techniques are complementary and when applied to the same mixture of compounds, a very different elution order and separation speed and efficiency can result.

[0003] Modern HPLC and SFC systems are modular with functions of pumping, sample injection, column thermal control and detection being organized in separate electronic modules collectively controlled and coordinated by a computer workstation. SFC systems typically adds control features including augmented control of the carbon dioxide pumping subsystem and backpressure regulation to the system in order to condition the CO2 flows and pressure to maintain a reproducible monophasic mobile phase in the separation flow path extending at least from the point of sample injection through detection. Both types of chromatography systems can typically be run in either isocratic or gradient elution modes. Isocratic elution mode occurs when the composition of the mobile phase is kept constant during the course of a separation. Gradient elution mode occurs when the composition is varied either continuously or by stepwise changes during the separation. Generally HPLC separations have a constant total flow rate, but some methods also vary flow during the separation. The total flow rates in HPLC are typically much lower than in SFC due the relatively higher viscosities and lower diffusion rates in HPLC mobile phases.

[0004] In the course of experimental synthesis of new compounds, as in the pharmaceutical industry, it is beneficial to periodically analyze reaction products either intermediately or at the end of synthesis process to determine the success of the synthesis or at other times during drug discovery and development. Such analyses are performed by chemists that are often not specialists in the use of chromatographic equipment and prefer significant automation in using the systems. In addition, the high cost of certain equipment such as mass spectrometers makes sharing of equipment a common practice in these industries. Since both HPLC and SFC are routinely used in these industries, it would be beneficial to allow users to easily and/or automatically convert between HPLC and SFC in a single instrument configuration. Several attempts to merge the capabilities of the two techniques have been reported, but in all cases, due to the customization typically associated with SFC related pumps, entirely different sets of pumps have been used.

[0005] A quick review of the different types of HPLC and SFC systems of prior art will assist in demonstrating common and opposing requirements of the prior art flow systems. Figure 1 illustrates the flow path of a modern binary HPLC system of prior art. Two high pressure metering pumps 100 and 1 10 are used to create the mobile phase from solvent reservoirs such as reservoir 120 plumbed to the inlet of each pump. High pressure mixing is performed at mixer 130 positioned at or after the flow junction of the two high pressure outlet flow streams. The mixed flow proceeds through the modular flow system through components typically including a sample injection module, a separation column typically housed in a thermal control module or column oven and a detector. The serial collection of modules is depicted as instrument cluster 140 in Figure 1 . Finally after exiting instrument cluster 140, flow is directed to a waste container 150. The flow system is capable of isocratic or gradient elution

chromatography for either reversed phase or normal phase HPLC; however normal phase HPLC is typically used only as a last resort owing to its very slow equilibration times and problems with reproducibility due to water adsorption. Not shown in Figure 1 are various optional components including column selection valves, multiple detectors, manual injection valves and other items that may be used to customize the system. Similar omission of optional components is common in all described flow paths.

[0006] Figure 2 represents an alternative HPLC flow system using a single quaternary pump 220 to dynamically formulate specific mobile phase compositions. Pump 220 uses a low pressure proportioning valve to draw simultaneously from a bank of four solvent reservoirs 230 in varying ratios. The aspirated mixture is drawn through the pump heads and typically a pulse damper and or internal mixer completes the mixing process of the mobile phase. The pump delivers to module cluster 140 and the flow stream terminates in HPLC waste vessel 150 as described earlier. Because the internal dwell volume of quaternary systems tends to be larger and the individual components of the mobile phase are aspirated rather than pumped by positive displacement, quaternary HPLC systems are typically not considered as precise as binary systems in very highly demanding applications. However, such limitation is typically taken into account during method development and extreme performance demands such as very fast gradients or ultra high pressures are typically avoided.

Under less stringent conditions, little difference is seen between low and high pressure mixing systems found in binary and quaternary system respectively. Like binary HPLC systems, quaternary flow systems are capable of isocratic or gradient elution

chromatography for either reversed phase or normal phase HPLC with the same performance limitations in normal phase.

[0007] Figure 3 illustrates a prior art SFC system. The system includes pump 1 10, high pressure mixer 130, and HPLC module cluster 140 which are essentially identical to those found in binary HPLC systems as illustrated in Figure 1 . For some types of HPLC modules, minor modifications are made such as converting injectors back to conventional sample loop injection modes or adapting detector cells for high pressure. New components in the SFC flow system include CO2 source 170, optional booster pump 180, CO2 metering pump 190, back pressure regulator (BPR) 200 and SFC waste container 210. The specialized equipment is required for the precise delivery of CO2 into the chromatographic system. Further, the mobile phase once mixed must be maintained at pressure generally above 80 bar to remain miscible.

Since these pressures would otherwise be lost after the separation column, BPR 200 is required. Finally, custom waste container 210 is required that receives both expanded CO2 vapor and liquid organic modifiers and collects the liquid phase prior to venting. pSFC flow systems of the configuration of Figure 3 are capable of isocratic or gradient elution chromatography generally for normal phase separations exclusively.

[0008] Highly pressurized CO2 is typically used in pSFC systems as the weak solvent for binary separations. Because of its high compressibility even in the liquid state, CO2 requires one or more flow enabling devices in order to be pumped precisely. Such flow enabling devices can include chillers, heat exchangers, booster pumps, and modified metering pumps as well as other devices. The requirements of booster pump 180 in Figure 3 are highly dependent on the type of CO2 source connected. Older commercial systems did not require any booster if the CO2 was delivered from a liquid eductor tube of a high pressure liquid CO2 cylinder. Metering pump 190 took full responsibility for chilling, compressing and metering the CO2 as a liquid. When source 170 was a cryogenic dewar or storage tank, a booster pump 180 typically in the form of an air driven gas booster was required to bring the pressure up to a constant state that required less than a few hundred watts of power near zero °C in order to bring the CO2 to a liquid state before pumping. Metering pump 190 remained responsible for final compression to the column pressure and CO2 metering functions.

[0009] A recent invention described by US patent application Serial Number 12/230,875 demonstrates an alternate use of booster pump 180. In this case, pump 180 is modified to provide both chilling and dynamic precompression to a vapor phase inlet stream of CO2 between 40 and 70 bar. The pump liquefies the vapor stream and pumps it to a control pressure value directed at the inlet of metering pump 190 in a serial plumbing arrangement. The dynamic precompression is controlled by sensing the outlet pressure of pump 190 and remaining just a few bars below the sensed pressure regardless of pressure changes at the head of the column due to gradient elution. The effect of the invention is to enable conventional HPLC pumps to meter CO2 without further compression and with very little pump noise.

[0010] The SFC flow system of Figure 3 is most similar to the binary HPLC system of Figure 1 . It uses high pressure mixing at mixer 130 to achieve precise composition in mobile phases. High pressure mixing is a requirement of SFC since CO2 at pressure less than approximately 80 bar are not miscible with significant amounts of organic liquid modifiers. Generally separations are carried out in SFC against column head pressures ranging from 100 bar to 400 bar, although with modern HPLC pumps, head pressures over 600 bar are achievable today. The generally high back pressure required by pSFC is typically not a problem since mobile phases using CO2 are generally between 3 and 20 times less viscous than aqueous mobile phases and so significantly less pressure drop occurs across the separation column.

However, the presence of a high back pressure BPR could be an impediment when considering for use in a common flow path for both rHPLC or pSFC. Standard HPLC systems tend to expend all their pressure capacity just in overcoming flow resistance in the separation column. [0011] A second difficulty lies with the specialization of CO2 boosters and pumps used in a dual mode system. It is obvious to one skilled in the art that the CO2 flow must be isolated from the main chromatographic flow path during rHPLC operation. Exposure of an HPLC mobile phase to pressurized CO2 would cause severe

outgassing that interferes with all optical and most other detectors. Modern CO2 pumping systems also typically store a charge of highly pressurized CO2 in a reservoir within the flow system. Draining the stored CO2 by a sudden or unrestricted release of pressure is undesirable. Most CO2 pumping systems take some time to recharge to a level that is suitable for stabile chromatography so it is of advantage to maintain the pumping system in a charged state.

[0012] As mentioned above, pSFC is typically considered a normal phase technique although niche applications have used C18 and cyano stationary phases with high polarity modifiers. Since CO2 at liquid-like densities has a polarity approximating hexane, it does not substantially dissolve water except as an additive to other more soluble organic modifiers. Nor do CO2 mobile phases tolerate ionized compounds, again except as supported by the polar organic modifiers used in the mobile phase. Further ionic species tend to accumulate strongly on the polar stationary phases and do not elute leading to loss of column efficiency and eventual flow blockage. This is in direct conflict with the use of ionized buffers in HPLC to control speciation of polar compounds. Significant care must be taken to rinse common flow paths in

chromatographic systems that can switch between pSFC and rHPLC applications to prevent outright precipitation of residual buffers in the flow lines or the columns due to mobile phase incompatibility.

[0013] There is an unmet demand for a method and device for rapidly switching between the rHPLC and pSFC modes in a common instrumentation configuration. The two techniques are complimentary with opposing separation mechanisms. Hence if a separation fails or is very long in one mode, it will likely succeed and or shorten in the other. Such a system further diminishes the concern of dedicating a system fully to a less familiar technique such as SFC for the main stream of users. Ideally, the method uses the same high pressure pumps for both modes of operation rather than different banks for each mode. Finally, there is a significant economic driver of being able to share expensive components such as mass spectrometers or electronic light scattering detectors (ELSD's) between modes, dramatically improving the confidence in the separation in both modes.

[0014] Care must be taken in any dual mode system to insure full conversion of flow paths between modes and adequate rinsing capability of common flow paths. Further, overall utility of the system requires that CO2 subsystems remain charged in a safe manner during rHPLC separations for rapid reconversion to SFC modes.

SUMMARY

[0015] Embodiments for an automated, reversible means of converting a standard, high performance liquid chromatography (HPLC) flow path to a flow path suitable for supercritical fluid chromatography (SFC) are described. The invention uses a device or process to selectively switch flowpaths to achieve different modes of operation such as HPLC and SFC. Preferably, a single valve, for example a high pressure rotary valve, is used to 1 ) assure all fluid lines are switched simultaneously, 2) share HPLC modules in both HPLC and SFC modes, 3) maintain a desired fluidic communication between the CO2 pumping subsystem and the system back pressure regulator and 4) provide an adequate means of rinsing the shared fluidic path of incompatible solvents prior to switching between flow paths. The invention is applied to a variety of flow configurations including binary, high pressure solvent mixing systems and quaternary, low pressure solvent mixing systems. The technique is generally applied to the fields of supercritical fluid chromatography and high pressure liquid chromatography, but users skilled in the art will find utility for any flow system where pressurization components must be periodically applied to and removed from both ends of a flow stream in an automated manner.

[0016] The various preferred and alterative embodiments of the present invention employ a high pressure rotary valve to achieve changes in the flow

configuration to convert a system between HPLC operation and SFC operation.

Conversions are performed with the following restrictions. The manufacturer's requirement for products that can be used in a commercial embodiment of an SFC booster pumping system products require that the CO2 booster and BPR be maintained in fluid communication at all times during operation of the system. The requirement is implemented to provide a safe, vented overpressurization path from the CO2 booster via the BPR and as a means of retaining the system in a charged and ready state while operating in alternate chromatographic modes. The created flow systems should as closely as possible represent the flow systems of the unmodified HPLC flow systems and the prior art or newly described SFC flow systems respectively as illustrated in figures 1 through 5. The flow system conversion should result from a single switching of a single rotary valve. Use of multiple valves for conversion makes fluidic system more complex an increase the risk of safety hazards if one valve switches and another fails to switch.

[0017] Various described embodiments further improve the art of SFC by combining programmable low pressure formulation of the organic modifier composition with high pressure mixing between the modifier and CO2. This provides the ability of on- demand selection of the strong solvent formulation within the mobile phase previously absent from commercial SFC systems. Such embodiments also realize certain limitations described herein as part of the novel use of quaternary pumps in SFC.

BRIEF DESCRIPTION OF FIGURES

[0018] The accompanying figures where like reference numerals refer to identical or functionally similar elements throughout the separate views and which together with the detailed description below are incorporated in an form part of the specification, serve to further illustrate various embodiments and to explain various principles and advantages:

Fig. 1 is a depiction of the flow path for a binary HPLC system of the prior art;

Fig. 2 is a depiction of the flow path of a quaternary HPLC system;

Fig. 3 is a depiction of the flow path of a binary SFC system of the prior art;

Figs. 4 A and 4B depict the two valve states of an 8-port valve arrangement

enabling conversion between binary HPLC and binary SFC; Figs. 5 A and 5B depict the two valve states of an 8-port valve arrangement enabling conversion between quaternary HPLC and binary SFC with custom modifier composition;

Fig. 6 is a depiction of the flow path of a modified binary SFC system using a quaternary pump to supply custom modifier compositions as in Fig 5B; Fig. 7 is a chart showing the effect of varying modifier composition on range of analytes separated by SFC;

Figs. 8 A and 8B depict the two valve states of an 8-port valve arrangement

enabling conversion between quaternary HPLC and modified binary SFC with custom modifier and premixing with a high pressure CO2 stream prior to a metering pump supplying accurate total flow;

Fig. 9 is a depiction of the flow path of a modified binary SFC system using a quaternary pump to supply custom modifier compositions and premixing with a high pressure CO2 stream prior to a metering pump supplying accurate total flow as in Fig 7B;

Figs. 10 A and 10B depict the two valve states of a 10-port valve arrangement enabling conversion between quaternary HPLC and modified binary SFC with custom modifier composition;

Figs. 11 A and 11 B depict the two valve states of a 10-port valve arrangement enabling conversion between quaternary HPLC and modified binary SFC with custom modifier composition and delivering flow to a mass spectrometer;

Figs. 12 A and B depict the two flow paths created by the valve arrangement of Figs 11 A and 11 B and delivering flow to a mass spectrometer.

DETAILED DESCRIPTION

[0019] In the following description of preferred and alternative embodiments, reference is made to the accompanying drawings which form a part hereof, and in which it is shown by way of illustration specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural or process changes may be made without departing from the scope of the preferred embodiments of the present invention. One skilled in the relevant art will recognize that many possible modifications and combinations of the disclosed embodiments can be used, while still employing the same basic underlying mechanisms and methodologies. The descriptions herein, for purposes of explanation, have been written with references to specific embodiments. However, the illustrative discussions within the present application are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed.

[0020] Preferred and exemplary embodiments of the invention applied to a binary HPLC pumping system are illustrated in Figures 4A and 4B. Functional system diagrams in Figures 4A and 4B display the two unique positions of 8-port switching valve 300. Fluidically connected valve positions 302 are numbered one through eight in the figures. Each figure shows 1 ) the valve arrangement connections to various modules of the HPLC or SFC flow system respectively; 2) the primary flow path demarked by flow arrows and 3) the secondary flow path or paths marked with dotted flow lines. Although variations of a multi-port selection valve are shown and described in the embodiments, one skilled in the art will appreciate that the device and process used to selective switch or change flow paths in the present invention is not limited to a multi- port valve or use of such a valve; other devices and processes could be arranged that can switch flowstreams such as automatic electronic switches, manual switches or valves without departing from the scope or intent of the claims of the present invention.

[0021] The system embodied in Figure 4A represents the valve position in its binary HPLC mode. In this valve position, solvent selection valve 310 is used as a liquid solvent inlet to pump 190 to provide both a variety of suitable solvents 330 for the HPLC process. One position of solvent selection valve 310 is reserved for connection to vent 320 which is used to drain the small volume of CO2 based mobile phase trapped in line 340 when valve 300 is switched. Venting is performed to insure solvents can be primed into the inlet of metering pump 190 with relative ease. Pump 190 is a

conventional HPLC or UHPLC pump modifier for high pressure input and capable of pumping either CO2 or HPLC type fluids. In this latter mode pump 190 is equivalent to pump 100 of Figure 1 . Similarly, Pump 1 10 typically will pump water during HPLC separations and is identical to the pump 1 10 of Figure 1 . In this configuration, HPLC mobile phases are created by mixing the outputs of pumps 190 and 1 10 through mixer 13; directing the mobile phase liquids through instrument cluster 140 which performs the HPLC separation and flowing on to HPLC waste 150 which accumulates the spent mobile phase. As a result, Figure 4A provides a fully implemented high pressure mixing binary HPLC mode similar to Figure 1 . Mixer 130 is comprised of mixing elements known in the art or anticipated devices which enable the mixing of two or more flow streams to near homogeneity within delay volumes practical for chromatography. It should be noted that the secondary flow path which includes CO2 source 170, CO2 booster 180, BPR 200, and SFC waste 201 maintains communication between the charges CO2 supply sub system and the vented BPR. As such, the supply subsystem may be maintained safely at full charge and ready to deliver when the SFC mode is engaged.

[0022] Switching valve 300 to its alternate position creates a new flow path configuration that enables an SFC mode of operation as illustrated in Figure 4B. In this configuration, the main flow path is suitable for binary SFC as depicted in Fig 3. CO2 booster pump 180 becomes the sole supply to metering pump 190 transforming this side of the pumping system for CO2 delivery. Metering pump 1 10 is switched to a suitable organic solvent modifier miscible with CO2 such as methanol. BPR 200 connects to the detector outlet from instrument cluster 140 and completes the

requirement for back pressure control. Selection valve 130 and HPLC waste container 150 as secondary paths are isolated by plugs 304 and 306 respectively to prevent uncontrolled siphoning while not in use.

[0023] Certain considerations must be taken when switching between valve positions of Figs 4A and 4B. When switching from HPLC mode to SFC mode, pump 1 10 will be pumping water and pump 190 will be pumping the solvent selected from valve 310. As a precaution, the entire HPLC flow system should be rinsed with a solvent compatible with both water and CO2 such an alcohol. Typically users will have prepared switchover methods in the controlling workstation to accomplish this task. In cases where aqueous buffers are used, a rinse with water first then organic solvent should be implemented. Hence at the switching point of the valve, the entire system is primed with compatible solvent. Also at this time it will likely be appropriate to select the column to be used in the SFC mode and rinse it with solvent as well. CO2 is pre- pressurized and is self priming. That is, the pressure of the CO2 from booster 180 is sufficient to force residual solvent from flow line 340 through pump 190 and allow pumping of CO2 from pump 190.

[0024] Switching valve 300 from SFC mode enables HPLC mode. First valve 310 is selects vent 320 as its flow path. Valve 300 is switched to the position in Fig 4A and residual pressurized CO2 is vented from flow line 340. Valve 310 the selects an appropriate rinsing solvent and Pump 190 is primed. Automatic priming may require use of an automated prime valve, not shown to allow the organic solvent to fill flow line 340. An appropriate HPLC column is selected and the flow system is primed with neutral organic solvent. Pump 1 10 is switched to water and then any specialized aqueous buffer and the system is ready for HPLC separations.

[0025] Figures 5A and 5B illustrate an alternate embodiment of the invention wherein an 8-port valve configuration allows switching between a quaternary HPLC flow path with low pressure mixing and a binary SFC flow path with low pressure mixing of modifiers and high pressure mixing of CO2 and modifier. In Fig 5A, the primary chromatographic flow path is highlighted with arrows. This flow path combines quaternary pump 220 with reservoirs 230 with instrument cluster 140 and HPLC waste container 150 exactly as described in Fig 2 above. Meanwhile the secondary flow path maintains CO2 metering pump in communication with BPR 200 in such a manner the CO2 pumping subsystem can remain charges with the BPR supplying a safe venting path against overpressurization.

[0026] Fig. 5B represents a new advance in the art of SFC. In this

configuration, the flow system resembles that of Figure 3 with simple high pressure mixing at mixer 130. A major difference, however, exists in this configuration. Figure 6 displays a schematic of the new flow path where quaternary pump 220 with reservoirs 230 replace the isocratic metering pump 1 10 if Fig 3. Hence, the user is able to select from a near infinite number of combinations of modifier compositions to combine with CO2 for the SFC separation. Figure 7 demonstrates the high importance of this feature. SFC is a dynamic art with continuous advances in its range of separation utility. Figure 7 shows a chart of the effective range of analysis of SFC in combination with varying compositions of modifiers. The original concept of SFC is shown first where pure CO2 was used as a modifier with a tunable solvating power based on the CO2 density. It was soon realized that addition of organic liquid modifiers dramatically extended the analyte range and shifted the emphasis from GC to LC like separations. Further extension of the techniques has been realized by addition of small quantities of acids or bases, called additives, the organic modifiers to control ionic speciation and compete with active sites on the stationary phase. Water has also been used as an additive typically to alcohol modifiers to enhance solubility of more polar species. The result of changing modifier composition has been to increase the effective range of SFC beyond that currently practiced by the sum of normal phase and reversed phase HPLC in total. As a result, an SFC system that can programmatically call up various combinations of modifiers and additives in varying compositions contributes greatly to the art.

[0027] Such an ability is not without limitations. As described earlier, quaternary pumps typically contain a large internal delay volume arising from internal pulse dampers and mixer elements not shown in Figure 6. Such delay volumes can range from hundreds to thousands of microliters. The presence of large delay volumes significantly separates the timing between changes in flow and changes of composition of flow coming from the quaternary pump. For example, if the delay volume were assumed to be 1000 uL, and the quaternary flow rate were 0.5 mL/min [500 uL/min] it would take 2 minutes before a change in the composition reached mixer 130. One the other hand, changes of total flow occur virtually instantly at the mixing point and at the same time alter the delay time between the modifier mixing point in the quaternary pump and mixer 130. In gradient elution chromatography, it is common to continuously chance the volumetric ratio of strong and weak solvents. In SFC this refers to modifier and CO2 respectively. This implies that typical SFC method will require a change in the flow rate coming from quaternary pump 220 during gradient elution. While the user may also wish to vary the modifier composition during this time, it is quite difficult, due to the large and variable time delay of the composition change in the flow system, to track what the flow and composition entering the column at any time. Conditions will also vary significantly as a function of the gradient rate and total SFC flow rates. As a result, users are not encouraged not to try varying both parameters. Instead the benefit of a single composition of modifier per run varying in flow rate remains very high, and allowing the composition to change between runs for subsequent analyses remains available.

[0028] Figures 8A and 8B illustrate another alternate embodiment of the invention wherein an 8-port valve configuration allows switching between a quaternary HPLC flow path with low pressure mixing and a binary SFC flow path with low pressure mixing of modifiers and high pressure mixing of CO2 and modifier. In this case, the high pressure mixing step occurs prior to pump 190 which now meters total flow rather than only CO2 flow. A description of this so-called quantitative solvation method of mixing has recently been submitted as a patent application to the US Patent office. In Fig 8A, as in figure 5a, the primary path is that of a quaternary HPLC of prior art, while the secondary path connects the CO2 pumping subsystem to the BPR and waste as described earlier.

[0029] Unique in this embodiment is the ability to switch to the flow configuration of Figure 8B which enjoys the advantages described for figure 5B when using a quaternary pump as the modifier generator as well as the stated limitations. In addition, the configuration allows the CO2 to go unmetered but rather simply to complete the total flow demand of metering pump 190 not delivered as modifier by quaternary pump 220. Figure 9 illustrates the details of the flow path. Booster 180 in this embodiment is of the type described by US patent application Serial Number 12/230,875. Booster pump 180 elevates the pressure of CO2 from CO2 source 170 to just below the outlet pressure of pump 190. Quaternary pump 220 delivers a flowstream less than or equal to the volumetric flow rate of metering pump 190 which determines total flow for the

chromatographic separation. When the volumetric flow of pump 220 matches the rate of pump 190, little or no CO2 is delivered to the final mobile phase except what may dissolve into the modifier under pressure. When the modifier flow is less than the total flow demand of pump 190, sufficient CO2 is delivered to complete the demand. Typical gradient runs in SFC general span compositions of 5% to 60% modifier. Thus in normal operation, CO2 volumetric delivery represents 40% to 95% of the total flow plus any CO2 required to make up for losses due to mixing. Flow continues through instrument cluster 140 and BPR 200 as stated in earlier configurations.

[0030] This arrangement has two major advantages. First, it provides a more controlled volumetric flow rate into instrument cluster 140 since volumetric losses from high pressure mixing in mixer 130 are made up by additional CO2. Second because booster 180 dynamically precompresses the mobile phase mixture before metering through metering pump 190, compressibility changes in the gradient mobile phase do not result in variable higher pump noise. Mobile phases generated by the configuration of Fig 8B are more compositionally accurate regarding the strong solvent of the separation.

[0031] Embodiments of the invention can be implemented by other than 8-port valves. Figures 10A and 10B illustrate an alternate embodiment of the invention wherein a 10-port valve configuration allows switching between a quaternary HPLC flow path with low pressure mixing and a binary SFC flow path with low pressure mixing of modifiers and high pressure mixing of CO2 and modifier. In these figures, the primary path is indicated by arrows and solid flow lines. The secondary path is indicated by dotted flow lines. Figures 10A and 10B add new elements of 10-port valve 400 and flow restrictor 410. The valve can be any 10 port high pressure switching valve with an upper pressure of at least 5000 psi. Flow restrictor 410 is typically a reduced id flow capillary with appropriate flexibility and length sufficient to connect to two adjacent ports of the valve. Restrictor 410 serves as a flow channel to complete the secondary fluidic path of Figure 10 A. It has the beneficial added use of providing a resistive diagnostic flow path for the CO2 delivery subsystem under simulated conditions of flow between pump 190 and BPR 200.

[0032] In Figure 10A, as in Figure 5A, the primary path is that of a quaternary HPLC of prior art, while the secondary path connects the CO2 pumping subsystem to the BPR 200 and waste container 210 as described earlier. The one difference as noted is the inclusion of restrictor 410 in the flow path which allows simulation of metered flow similar to flow through a column for diagnostic purposes. Figure 10B recreates the SFC mode flow path illustrated in figure 9 which has already been described. In this flow path, restrictor 410 is isolated from the rest of the flow stream. Pressure sensor 420 is included in the flow stream as an exemplary means of creating the pressure signal used by booster 180 in certain configurations of the CO2 pumping subsystem. The positioning of pressure sensor 420 is also exemplary as it could be located at any point along the flow stream prior to instrument cluster 140 so long as an insignificant pressure drop occurs between the pump outlet and the sensing point. Positioning in the pure CO2 flow stream is preferred, however since CO2 is less corrosive than some organic modifiers and additives used in SFC.

[0033] Figures 1 1 A and 1 1 B illustrate an alternate embodiment of the invention wherein a 2-position, 10-port valve configuration allows switching between a quaternary HPLC flow path with low pressure mixing and a binary SFC flow path with low pressure mixing of modifiers and high pressure mixing of CO2 and modifier and where the system delivers terminal flow to a mass spectrometer (MS). Figure 1 1A creates a flow path similar to the prior art flow path of Figure 2. However, rather than terminating in HPLC waste container 150, flow from the system terminates in mass spectrometer 440 which can typically accept flows of 0.05 to 2 mL/min of liquid flow. Secondary flow of Fig 1 1 A is illustrated as dashed lines. Valve 400 isolates the entire Cos pumping system 170, 180 and 190 , BPR 200 and SFC waste 210. Flow through the modifier inlet to mixer 140 is halted by plug 304. Metering pump 190 maintains communication with BPR 200 and SFC Waste 210 allowing it to remain safely charged. A schematic of the New quaternary HPLC/MS flow path is shown in Figure 12A.

[0034] Switching valve 400 to its alternate position creates the valve

configuration for the SFC/MS mode illustrated in Fig 1 1 B. In the primary path fluid exits pumps 190 and 220 and is combined in Mixer 130. Fluid is directed through instrument cluster 140 which may include a non destructive detector such as UV and then flows to BPR 200. After BPR 200, CO2 expands as much as 500 fold and only the organic modifier must be evaporated by the mass spec. However, the evaporating CO2 does place a significant heating load in the MS inlet and auxiliary heating may be required to supplement heating system of mass spec 440. In the secondary path of Fig 1 1 B, the SFC Waste container 210 is isolated from the flow system. Hence waste container 210 has only the function of providing a vented relief to the secondary path of Fig 1 1 A. A schematic of the new SFC/MS flow path is shown in Figure 12B. [0035] All the various embodiments described are exemplary and do not constitute a full listing on possible implementations of the invention. Extensibility has been show in the use of 8 and 10 port valves, but 12-port or higher valves may also be considered. Rotary valves may be replaces with appropriate combinations of normally open and normally closed solenoids, but this is less preferred due to the complexity and loss of robustness. The principle components of the invention include 1 ) the ability to switch reversibly and in a single step all fluidic flow lines of an HPLC system to convert the flow system for SFC use and back, 2) reuse of at least one high pressure metering pumps in both flow modes of each configuration; 3) maintenance of fluidic

communication between CO2 supply subsystem and the BPR in all modes of operation and 4) providing adequate ability to rinse common pathways with solvents miscible in both mobile phases for conversion between modes.

[0036] While this specification contains many specifics, these should not be construed as limitations on the scope of what is being claimed or of what may be claimed, but rather as descriptions of features specific to particular embodiments.

Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment.

Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a

subcombination.

Claims

CLAIMS What Is Claimed Is:

1 . An apparatus, comprising:

a fluidic switch that provides at least two positions where each position creates one primary and at least one secondary flow path configuration; and

a back pressure regulation module,

wherein the switch can be moved to a first position to select a first primary flow path configuration which enables a liquid chromatographic mode and to a second position to select a second primary flow path configuration which enables a supercritical fluid chromatographic mode, and

the back pressure regulation module communicates with the at least one secondary flow path when the liquid chromatographic mode is selected and with the primary flow path when the supercritical fluid chromatographic mode is selected.

2. The apparatus of claim 1 , further comprising:

a flow enabling module for compressible fluids,

wherein the switch provides the capability to maintain fluidic communication between said flow enabling module and said backpressure regulation module in both first and second said switch positions.

3. The apparatus of claim 2, wherein said flow enabling module is a C02 metering pump.

4. The apparatus of claim 2, wherein said flow enabling module is a C02 booster pump.

5. The apparatus of claim 1 or 2, further comprising:

an HPLC pump and a collection of HPLC modules modified as necessary for dual use within either a compressible fluid flow system or a liquid flow system,

wherein said HPLC pump and modules remain in the primary flow path configuration in both the first and second positions of said switch.

6. The apparatus of claim 5, wherein said HPLC pump is a high pressure positive displacement pump.

7. The apparatus of claim 6, wherein said high pressure positive

displacement pump is one of a quaternary pump module, an isocratic pump module, or one unit of a binary pump module.

8. The apparatus of claim 5, wherein at least one pump in each primary flow path configuration is capable of rinsing the flow path with a mobile phase compatible with both chromatographic modes of operation.

9. The apparatus of claim 5, wherein the first and the second primary flow path configurations each terminate through a mass spectrometer.

10. A system, comprising:

a liquid chromatography (LC) system of modules; and

a collection of supercritical fluid chromatography (SFC) modules, connected to the LC system of modules, the modules including:

a fluidic switch,

a back pressure regulator, and

a pump for compressible fluids,

wherein the switch can be selectively positioned to convert a flowpath between an LC system mode of operation and an SFC system mode of operation, and

fluidic communication is maintained between the said pump and said backpressure regulator module in both said modes of operation.

11 . The system in claim 10, wherein the LC system modules are modified as necessary for dual use within either a compressible fluid flow system or a liquid flow system and are included in the flow path of each mode of operation.

12. The system of claim 10, wherein at least one pump in each mode of operation is capable of rinsing the flow path with a mobile phase compatible with both chromatographic modes of operation.

13. The system of claim 10, wherein the LC system of modules further comprises:

a mass spectrometer, wherein each mode of operation is fluidically connected to said mass spectrometer.

14. The system of claim 10, wherein the LC system of modules includes a high performance liquid chromatography system of modules.

15. The system of claim 10, wherein the LC system of modules includes an ultra high performance liquid chromatography system of modules.

16. The system of claim 10, wherein the LC system of modules includes modules capable of operating within system pressures up to 600 bar.

17. A process, comprising:

selectively switching between a liquid chromatography (LC) flow path and an SFC flow path comprising an LC pump, chromatographic system

components, an upstream LC solvent source , an SFC compressible fluid mobile phase source, and SFC system components; and

converting a flowpath, between an LC mode of operation to an SFC mode of operation,

wherein fluidic communication is maintained between the SFC compressible fluid phase source and a back pressure regulator comprised within said SFC system components.

18. The process of claim 17, further comprising:

rinsing a flowpath to a state compatible with both modes of operation from either mode of operation.

19. The process of claim 17, further comprising:

directing flow of each mode of operation to a mass spectrometer.

20. The process of claim 17, wherein the step of selectively switching the LC flow path comprises selectively switching a high performance liquid

chromatography flow path or an ultra high performance liquid chromatography flow path.