APPLICATIONS OF MICROWAVE RADIATION TO CHROMATOGRAPHIC SEPARATIONS

Abstract

A method of applying microwave radiation to a chromatography column to achieve turbulent chromatography, increase the speed of column equilibration following a change of mobile phase, facilitate faster mixing of fluids, reduce the viscosity of fluids, and apply temperature pulses to selected time segments of a separation. The method may be used in conjunction with the use of columns packed with fused-core particles or monolithic columns. Improved size-based separations may be achieved using diffusion-and-turbulent-dependent size exclusion chromatography.

Claims

1. A method of enhancing turbulent flow chromatography in liquid or supercritical fluid separations for both retained and unretained solutes, comprising the step of applying microwave radiation to the chromatography column so as to enhance the molecular motion of the mobile phase molecules, lower the viscosity of the mobile phase, cause increased molecular motion of the analyte molecules within the mobile phase, minimize the residual laminar layer within a turbulent mobile phase, and increase the molecular motion of analytes within the stationary phase.

2. The method of claim 1, wherein the direction of the separation is not inconsistent with turbulent chromatography.

3. The method of claim 1, further including the step of using a fused core column.

4. The method of claim 1, further including the step of using a monolithic column.

5. The method of claim 1, further including the step of using a high flow rate.

6. The method of claim 5, wherein the mobile phase includes polar liquids.

7. The method of claim 1, further including the step of separating molecules according to their size using classical size exclusion chromatography.

8. The method of claim 7, wherein the size exclusion step is accomplished using a column composed of pores that allow only molecules below a certain molecular weight to enter and the separation is run at a linear velocity sufficiently high to induce turbulent flow.

9. The method of claim 1, wherein the microwave radiation enhances turbulent flow conditions already present in the column at a lower flow rate.

11. The method of claim 1, wherein the step of applying microwave radiation is performed only during the intervals within a separation where peak-free regions of the chromatogram are being eluted.

12. The method of claim 1, wherein the step of applying microwave radiation is performed only when non-critical peak pairs are being eluted.

13. The method of claim 1, wherein the chromatography column and the stationary phase are constructed of materials that do not absorb microwave radiation, wherein the microwave radiation directly heats only the mobile phase and analytes but not the column and any temperature elevation to the column is due to heat convectively transferred to the column housing and stationary phase particles.

14. An enthalpic method of enhancing turbulent column chromatography, comprising the steps of: providing a fused core or monolithic column; applying microwave radiation to the column; and eluting analytes in reverse order of molecular weight, such that larger molecules elute earlier than smaller molecules.

15. The method of claim 14, wherein a high porosity monolithic column is employed.

16. The method of claim 14, wherein the step of applying microwave radiation does not form a radial temperature gradient in the column.

17. A method of enhancing turbulent column chromatography, comprising the step of applying microwave radiation to a chromatography column following a change of the mobile phase in a re-equilibration stage of a gradient run in either liquid or supercritical fluid chromatography so as to increase the rate of mass transfer and the speed of column equilibration.

18. A method of enhancing turbulent column chromatography, comprising the step of applying microwave radiation to a chromatography column to increase the speed at which two or more liquid or supercritical phases mix uniformly.

20. The method of claim 19, further including the steps of: using carbon dioxide as the primary mobile phase; and adding a polar liquid modifier to change the polarity or density of the mobile phase, or to interact with and pacify active sites on the column.

Description

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0049] The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes reference to the annexed drawings wherein:

[0050] FIG. 1 is a highly schematic diagram showing a packed chromatography column with particle diameter, internal column diameter, and column length indicated.

[0051] FIG. 2 is a schematic view showing the difference between laminar and turbulent flow.

[0052] FIG. 3 is a highly schematic view illustrating a laminar flow profile.

[0053] FIG. 4 is a highly schematic view showing two molecules traveling under laminar conditions in a column.

[0054] FIG. 5 is a schematic diagram showing the relatively flat flow profile obtained under turbulent flow conditions.

[0055] FIG. 6 is a schematic diagram showing two molecules traveling under turbulent flow conditions.

[0056] FIG. 7 is a schematic view showing laminar and turbulent mobile phase regions resulting under turbulent flow conditions.

[0057] FIG. 8 is a graph illustrating the recoveries of lysozyme and parabens as a function of flow rate.

[0058] FIG. 9 is a graph illustrating the percentage removal of plasma proteins as a function of Reynolds number.

[0059] FIG. 10 is a graph illustrating the reduced plate height vs linear velocity curves for lysozyme and bovine serum albumin.

[0060] FIG. 11 is a graph illustrating small and large molecules with respect to the laminar and turbulent regions of the mobile phase.

[0061] FIG. 12 is a graph illustrating the phase diagram of carbon dioxide.

[0062] FIG. 13 is a graph illustrating the prior art: viz., the sequential elution of -chymotrypsinogen and lysozyme via step gradients on a cation exchange column.

[0063] FIG. 14 is a table showing the molecular weight range of proteins removed as a function of flow rate.

DETAILED DESCRIPTION OF THE INVENTION

First Embodiment: Microwave Enhanced Turbulent Chromatography

[0064] In what follows, it will be argued that successful turbulent chromatography can be obtained by applying microwave radiation, and when using a mode of separation where the direction of the separation is not inconsistent with that of turbulent chromatography (i.e., where large molecules elute first). In addition, the use of fused core or monolithic columns, in combination with these circumstances, is the best chance for achieving successful turbulent separations. As will be described in the next section, the microwave radiation serves two functions: the first is to minimize or reduce the residual laminar layer that exists within a turbulent mobile phase; and the second is to increase the molecular motion of the analytes within the stationary phase. In this way, it helps to address both of the fundamental issues that have impeded successful turbulent chromatography, historically.

[0065] Benefits of Microwave Radiation for Turbulent Separations:

[0066] As noted in the Background Discussion, above, there are two reasons that successful applications of turbulent chromatography have been limited to unretained or very weakly retained analytes. The first is that the benefits of turbulent flow do not extend to the stationary phase, and especially to the stationary phase located within the pores of the column. The mass transfer in the stationary phase is still a slow, diffusion-driven, process; and, therefore, there is significant band broadening due to the stationary phase mass transfer mechanism. The second is that it has not been possible to obtain a completely turbulent mobile phase.

[0067] In an embodiment of the present invention, denominated a first only to differentiate it from other embodiments but not otherwise signifying relative importance, microwave radiation is used to address these problems and, therefore, to allow the benefits of turbulent flow to be fully realized for liquid or supercritical fluid separations, for both retained and unretained solutes. This is something which has previously not been possible. Furthermore, the first embodiment of this application will introduce a chromatographic mode of separation that has not previously been used.

[0068] To accomplish microwave enhanced turbulent chromatography (METC) one conducts a liquid or supercritical fluid separation under conditions so as to induce turbulent flow, such as high flow rates and large diameter particles which have a rough surface; while simultaneously, applying microwave radiation to the column. When microwave radiation is applied to polar liquids, a substantial degree of molecular motion is generated due, primarily, to the dielectric polarization mechanism. This is the same phenomenon that results in heating of polar liquids by microwave radiation. The energy that is delivered by this effect is substantial such that, for example, water in a small test tube will boil in less than 10 seconds. When the radiation is applied to a chromatographic system, one effect may be to enhance the molecular motion of the mobile phase molecules which will, in turn, cause increased molecular motion of the analyte molecules. And, when dealing with analytes having some polarity, the radiation may have a direct effect on the analytes as well. The increased molecular motion that results from these effects is particularly important with respect to the liquid in the pores of the column. As stated previously, there is only slow diffusion-driven motion in the pores, as the benefits of turbulent flow are only realized for the flowing portion of the mobile phase. Therefore, there is a significant band broadening contribution due to the stationary phase mass transfer mechanism, in the absence of microwave radiation.

[0069] To further minimize the concern of stationary phase mass transfer, use may also be made of a column composed of pellicular (or fused core) particles where only the outer portion of the particle is porous. It is well known that such particles reduce the stationary phase mass transfer contribution to band broadening, especially for large molecules. Use of monolithic columns may also be an option provided that turbulent flow can be readily achieved, and it may be beneficial to construct a monolith of higher than normal porosity for this purpose. Non-porous or open-tubular columns may be an option, in certain situation, although there may be a significant loss of loadability and retention with such columns.

[0070] A second advantage in using microwave energy is to create a substantial mixing effect which results in the laminar layer mixing into the bulk turbulent layer, such that a thinner laminar layer results. Or, optimally, the laminar layer is entirely mixed into the turbulent mobile phase such that one continuous, turbulent, mobile phase is obtained. By virtue of this effect, the second limitation in successfully utilizing turbulent chromatographythe detrimental effect of the residual laminar layeris addressed. However, and reiterating an earlier note above, the second of the more recent insights into turbulent chromatography is that large molecules realize a greater reduction in mobile phase mass transfer than small molecules. The present inventor believes this is due to the fact that they cannot fully enter the thin laminar layer (see FIG. 11). Thus, for large molecules it is expected that this would not be a concern, though it is not yet known how large a molecule must be for this to be realized. However, the data shown in FIG. 10 suggest it may be somewhere between 13,000 and 64,000 Da.

[0071] There are further advantages of microwave enhanced turbulent chromatography over classical turbulent chromatography. First, the microwave radiation further increases the mass transfer of the solutes in the mobile phase, above and beyond the increase due to the turbulent flow. Secondly, the molecular motion caused by the radiation results in the onset of turbulent flow at lower flow rates than that usually observed, or what would be predicted by Reynolds number calculations. There are several advantages to running at a lower flow rate than typically used in turbulent chromatography; ideally, the lowest flow rate at which the flow is sufficiently turbulent to accomplish the fundamental goal of minimizing mobile phase mass transfer. First, these lower flow rates are more easily handled by chromatographic systems. Second, the lower flow rates further minimize the extent to which stationary phase mass transfer effects will be observed. And lastly, the lower flow rates are more efficient in a given length of column. To elaborate on the last point, it has been realized that the very high flow rates at which turbulent chromatography is generally conducted would require the use of longer columns (Pretorius and Smutz, 1966). This is because at higher flow rates, the analytes travel a longer distance between the time they enter the mobile phase and the time they return to the stationary phase. Therefore, the analytes experience less of the stationary phase. One may say that the effective length of the column becomes shorter as the flow rate increases beyond a certain point. In addition, the data presented above suggest that under turbulent conditions there is an additional mechanism that reduces the access of large molecules to the stationary phase, suggesting that the issue of efficiency for a given length of column is even more significant when dealing with large molecules under turbulent conditions. (It may be noted that this flow-rate-dependent access to the stationary phase exists even for conventional chromatography, yet there is nothing in the fundamental theories of chromatography to address this. One might consider whether there is any value in adding a factor to the fundamental resolution equation to adjust the length term with respect to the linear velocity at which one is running). It should be clarified that the flow rate used for microwave enhanced turbulent separations will still be considerably higher than for a conventional HPLC method. Therefore, a compromise is reached between obtaining the desired faster separations and minimizing both stationary phase mass transfer issues and the need to use excessively long columns.

[0072] The benefits of turbulent flow apply not only to the chromatographic separation; turbulent flow is also known to minimize broadening of peaks as they move through the tubing, connections and the detector cell, i.e., band broadening due to extra-column effects (De Pauw, Choikhet, Desmet, Broeckhoven, 2014; Berger 2011). Therefore, this should be considered in the overall system design.

[0073] It was mentioned that the dielectric polarization phenomenon affects polar molecules. Thus, for separations that make use of non-polar mobile phases, such as carbon dioxide, some percentage of polar modifier must be added to the mobile phase to observe these benefits, unless there is sufficient direct interaction of the radiation with the analytes. However, such separations are, in fact, usually run with some polar modifier present.

[0074] A New Chromatographic Mode: Diffusion-and-Turbulent-Dependent Size Exclusion-Chromatography (DT-SEC):

[0075] One of the more recent insights into the nature of turbulent chromatography, covered in the Background Discussion above, is that under turbulent conditions the retention of large molecules decreases. This phenomenon has been used for sample preparation to accomplish the removal of large molecules (as with the commercially available TurboFlow columns). However, use of this size selective mechanism for chromatographic purposes has not yet been reported. Such a novel method would therefore be a unique mode of separation. The method would preferably make use of a column with pores large enough to allow unhindered diffusion of all molecules being separated. Large molecules would elute earlier than small molecules as they would have less access to the stationary phase due to the diffusional and turbulent processes described previously. The column would most likely consist of relatively large diameter particles (50 m, for example) and would be operated at high enough linear velocity so that turbulent flow would be achieved. In addition, it may be beneficial if the particles are of sufficiently strong construction so as not to deform during the separation. Just as with classical size exclusion chromatography, the conditions of the method should be chosen so as to minimize enthalpic interactions of the analytes with the column. It is envisioned that this method would be most useful for the separation of large molecules (i.e., the separation of large from larger from larger still). However, the method may have some usefulness for small molecules as well, and this would need to be evaluated experimentally. Given that this method would make use of the size selective retention characteristics due to both differences in diffusion as a function of the size of the analyte molecules as well as the differences in the way in which turbulent flow interacts with molecules as a function of size, it is aptly termed Diffusion-and-Turbulent-Dependent Size Exclusion Chromatography (or DT-SEC). It is interesting to note that with this technique, the flow rate at which the column is operated becomes a selectivity parameter that may affect the spacing of the peaks, which is not usually the case with isocratic separations. Furthermore, diffusional factors affect selectivity with this technique, whereas in other modes of chromatography diffusional factors are thought to affect only efficiency. When microwave radiation is used in combination with DT-SEC the technique may be given the name MDT-SEC (Microwave-Enhanced-Diffusion-and-Turbulent-Dependent Size Exclusion Chromatography).

[0076] Classical Modes of Chromatography that are Amenable to Use of Turbulent Conditions:

[0077] Keeping in mind one of the newer insights, considered in the Background Discussion above, that under turbulent conditions, there is a mechanism that results in retention being an inverse function of molecular weight, we may now revisit the question of which of the classical modes of chromatography could successfully exploit the benefits of turbulent chromatography. In many enthalpic separations, especially reversed phase separations, smaller analytes typically elute earlier than larger analytes. This is opposite to the diffusion-turbulent based mechanism, and therefore such methods are expected to be problematic under turbulent conditions, as the two modes of separation would work against one another. However, in any separation where larger analytes elute earlier than smaller analytes, turbulent chromatography could be successfully used. It is also expected that turbulent flow could be used successfully, with enthalpic methods, in cases where the analytes are not significantly different in molecular weight or when the differences are such that the effect of the diffusion-turbulent mechanism is not significant (in other words, the effect on retention is approximately equivalent for all analytes). Additionally, turbulent chromatography could be exploited for separations which elute one molecule at a time. In fact, an example of this was reported by Quinn et. al. (U.S. Pat. No. 5,795,469) where -Chymotrypsinogen and Lysozyme were sequentially eluted from a cation exchange column with a step gradient: 0.2 M NaCl in the first step, and 2 M NaCl in the second. The resulting separation 130 is presented in FIG. 13. It may be noted that the run time was fast, the peaks were baseline resolved, and the peak widths were 4 to 5 seconds, suggesting the potential for very high efficiencies and speeds. It should also be reiterated that the flat flow profile that occurs under turbulent flow conditions makes these type of step gradients more feasible.

[0078] It is often the case with one-molecule-at-time methods that each analyte is minimally retained as it is eluting, and under those conditions, the benefits of turbulence would be observed, even in the absence of microwave radiation. This approach is probably more applicable to large molecules, due to the on/off mechanism by which they separate (or, alternatively, due to their high S values).

[0079] Lastly, it will be noted that the DT-SEC or MDT-SEC mechanism could be used in combination with classical size exclusion chromatography, if using a column that were composed of pores that only allowed molecules below a certain molecular weight to enter (as with a conventional SEC column), but where the particles are of larger diameter and sufficiently strong, and if the separation were run at a high enough linear velocity to induce turbulent flow.

[0080] In summary, the three keys to successful turbulent chromatography with enthalpic methods include:

[0081] 1) Working with an enthalpic mode in which the analytes elute in reverse order of molecular weight (i.e., larger molecules elute earlier), or where the analytes are not significantly different in molecular weight, such that the diffusion-turbulent mechanism has an insignificant effect on the separation, or for methods where one molecule is eluting at a time. In the first case, the separation would be a function of the enthalpic mode as well as the diffusion-turbulent mechanism, as both factors have an effect on the extent of interaction of the analyte molecules with the stationary phase.

[0082] 2) Applying microwave radiation. In most cases, it is suggested that this should be done in such a way as to avoid significant heating, so that a radial temperature gradient does not form in the column.

[0083] 3) Using a fused core or monolithic column. High porosity monoliths may be ideal. Non-porous or open-tubular columns may be an option in certain situations, although there may be a significant loss of loadability and retention with such columns. Successful turbulent chromatography may be possible without microwave radiation in the following cases: a) for one-molecule-at-a-time methods where each analyte is minimally retained as it elutes; and b) when dealing with large molecules and using an open-tubular or non-porous column (assuming the method being used is consistent with the requirements of key 1 above). It is possible that some success could also be achieved with fused core or monolithic columns.

[0084] It may be noted from the discussion thus far that supercritical fluid chromatography (SFC) is, in some ways, particularly amenable to being used under turbulent conditions. Because the viscosity is lower, we expect (from equations 1 and 2) that turbulent flow would be more readily achieved at lower flow rates, which is beneficial in several respects as discussed above. And, as will be discussed with respect to a third embodiment of the inventive method, the application of microwave energy may also make it more feasible to use water as the primary modifier in an SFC separation.

[0085] It was suggested in U.S. Pat. No. 6,630,354 B2 that microwave radiation should be applied at lower powers and/or with short pulses, with intervals of rest in between, so as to avoid significant heating of the mobile phase. The reason is that if the temperature of the mobile phase is elevated significantly, a radial temperature gradient develops that will be harmful to the separation. However, a greater degree of energy can be applied when working with a turbulent chromatography system. There are two reasons for this: First, the higher linear velocities at which one is working means there will be less time for heat to accumulate. Second, the enhanced radial mass transfer means that the effect of any radial temperature gradient should be less detrimental as they effects would be averaged out, at least partly, as described in the discussion relating to FIG. 6.

[0086] Accomplishing Easier On-line Removal of Large Molecules:

[0087] In U.S. Pat. No. 9,804,133, to Stone, and co-pending U.S. patent application Ser. No. 15/400,473 (each incorporated in their entirety by reference herein), many compelling applications for the use of pre-columns are discussed. Any of these applications could be used similarly except where the columns would be designed to promote turbulent flow and would be operated under turbulent condition.

[0088] A particularly compelling application would be use of a pre-column followed by an analytical column to allow an easier alternative to the current approach for using turbulent flow to accomplish on-line removal of large molecule interferences. Such systems are generally designed with a valve which diverts the large molecules to waste during the first step of the process, and which is then switched such that the small molecules can be eluted and analyzed. An example of this would be the TurboFlow columns described earlier. In what follows the term TurboFlow column will be used to refer to the column that serves to rapidly elute large molecules by virtue of the turbulent conditions that it generates, with the understanding that this would not necessarily be the commercial product known as a TurboFlow column, but could be any column which serves this function.

[0089] The complication with the approach described above is that the small molecules elute from the TurboFlow column with poor peak shapes (for all the reasons described earlier with respect to why turbulent flow chromatography has not been successful historically). Therefore, a second column (the analytical column) is usually needed to re-focus and then separate the small molecule analytes. Since a chromatographically strong solvent is used to elute the analytes from the TurboFlow column, poor focusing would be obtained if the sample were transferred directly to the analytical column. The typical solution is to combine this elution flow stream with another flow stream of chromatographically weak solvent, wherein, the flow from the chromatographically weak fluid line must be higher than that from the chromatographically strong fluid line to render the resulting (combined) mobile phase as chromatographically weak. In this way, efficient focusing of the analytes may occur at the head of the analytical column (Edge, 2003; Chassaing, Robinson, 2009; Herman, Edge, 2012; Crouchman, 2012).

[0090] Given the complex nature of this process, additional method development is required such that the flow rates and chromatographic strengths of the two fluid streams are properly balanced so as to allow the small molecules to be efficiently transferred from the TurboFlow column and focused at the head of the analytical column. Additionally, such setups generally require a specialized and dedicated instrument with two pumps and the necessary valving (Edge, 2003; Chassaing, Robinson, 2009; Herman, Edge, 2012; Crouchman, 2012). [0105]

[0091] The successful application of turbulent chromatography, as described in this application, would make it possible to use one column, operated under turbulent conditions, to accomplish both the removal of large molecules in the first step, and the subsequent chromatographic separation of the small molecules. While such an approach could work, it is likely that there would be one remaining problem with this type of setup. Although the small molecules would be retained during the first step of the process, they are likely to be somewhat spread out on the column and, therefore, less than optimal efficiency would result in the subsequent chromatographic separation.

[0092] A simple solution to this problem would be to utilize a setup with two columns in tandem: the first serving as both the TurboFlow column and as a pre-column, and the second being the analytical column. With this setup it would only be necessary for the flow to be turbulent during the initial loading step, as it is the nature of turbulent flow that results in reduced retention for the large molecules. During the subsequent transfer of analytes from the TurboFlow/pre-column to the analytical column, as well as during the separation on the analytical column, the flow could be either turbulent or laminar.

[0093] In order for the desired focusing to be accomplished, at the head of the analytical column, the linear velocity of the analytes on the TurboFlow/pre-column must be notably higher than their linear velocity on the analytical column, during the transfer. This was discussed in detail in the patents mentioned above. One scenario by which this could be accomplished would be if the TurboFlow/pre-column was less retentive than the analytical column (with the same mode of chromatography occurring on both columns). For example, if doing reversed phase chromatography, and the TurboFlow/pre-column is composed of a cyano phase and the analytical column is composed of a C18 phase. One may also consider using a large pore or non-porous column; or a high porosity monolith or an open-tubular column (including a support coated open-tubular columns) as the TurboFlow/pre-column as these type of column formats also serve to lower the retention of analytes. In addition, it may be beneficial if the internal diameter of the TurboFlow/pre-column were narrower than that of the analytical column, as this would further promote a higher linear velocity on the TurboFlow/pre-column.

[0094] A valve may be placed in between the pre-column and the analytical column in order to direct the large molecules to waste during the first step. Alternatively, the valve may be located after both columns. And, in fact, one may choose to use no valve at all (if there is no concern with respect to the large molecules entering the detector). Though, in this case, the analytical column would need to tolerate the high flow rates used during the loading step. Therefore, if non-turbulent conditions are being used for the analytical separation, a monolithic column may be a good choice.

[0095] It may be noted that the increased ability to use step gradients and fast gradients, under turbulent conditions, may further enable the effective use of this approach, as it may be desirable to utilize a step gradient to transfer the analytes from the TurboFlow/pre-column to the analytical column. And fast gradients are almost always desired for bioanalytical separations. This approach would allow a far more simple approach in comparison to the historical approach, described above, which requires additional method development and specialized and dedicated instrument with two pumps and valving. Given its somewhat reduced retention, the TurboFlow/pre-column may need to be longer than usual.

[0096] Several additional embodiments will now be discussed; each of which could be utilized under either turbulent of laminar conditions.

Second EmbodimentUse of Microwave Radiation to Increase the Speed of Column Equilibration Following a Change of Mobile Phase

[0097] The second embodiment of the invention applies to the equilibration of a column after a change of mobile phase, e.g., the re-equilibration stage of a gradient run in either liquid or supercritical fluid chromatography. The process of equilibration is generally thought to require 10 column volumes or more, and therefore, requires a certain degree of time which adds to the overall analysis time. Researchers have shown recently that the process of equilibration can be as much as 65% diffusion controlled (J. Foley, 2014; J. Dolan 2015). This means that increasing the flow rate would have only a moderate effect on the rate of column equilibration (and would also result in more consumption of mobile phase, which is more costly and less environmentally friendly). By application of microwave radiation, the rate of mass transfer is increased, and therefore, the rate at which column equilibration occurs is accelerated.

Third EmbodimentUse of Microwave Radiation to Increase the Speed at which Liquid or Supercritical Phases Mix Uniformly

[0098] The third embodiment of the invention is the use of microwave radiation to speed the process of two or more liquid or supercritical phases mixing with one another. It is known that when two different phases begin to mix, even when the phases are entirely miscible with one another, complete and uniform mixing does not occur immediately. As a result, when two phases are mixed together during a chromatographic separation there is an interval of time where the mobile phase will contain packets of one phase and packets of the other. And this has a negative effect on the chromatographic separation as some solutes experience a different mobile phase then other solutes, as they pass through the column. This phenomenon exists not just in binary systems but for ternary (containing three components) or higher order systems as well. Furthermore, in cases where the phases are marginally soluble with one another, the rate of mixing is expected to be even slower and the potential exists for stratification to occur even after the solvents have mixed.

[0099] A particularly noteworthy example of the difficulties that can result from this type of issue is Supercritical or Subcritical Fluid Chromatography, in cases where carbon dioxide is used as the primary mobile phase and one desires to add some percentage of a polar liquid (or modifier) to change the polarity or density of the mobile phase, or to interact with and pacify active sites on the column. Water has been shown to be a beneficial modifier. However, water is only soluble to the extent of less than 3% in carbon dioxide (with the exact percentage varying depending on the temperature and pressure being used). As a result of the limited solubility, most SFC separations use other modifiers such as methanol or isopropyl alcohol. However, there would be benefits to using water and carbon dioxide, either alone, or with only very minimal quantities of another modifier present. Firstly, with only water and carbon dioxide, or with very minimal quantities of another modifier present, it would be possible to use a flame ionization detector (FID). This is quite desirable as the FID detector has many positive attributes such as good sensitivity, a wide linear range, and essentially uniform response factors for most solutes. Generally, an FID detector is not useable with liquid or supercritical fluid separations. However, under these conditions it would be, as both carbon dioxide and water give virtually no signal with the FID and, therefore, would not contribute a significant background response. Secondly, water and carbon dioxide are very environmentally friendly solvents. The high degree of mass transfer and mixing energy that results from the application of microwave radiation would make the use of water as a modifier more feasible, such that these techniques could be conducted without the presence of an additional modifier, or with a very small quantity of additional modifier present.

Fourth EmbodimentUse of Microwave Radiation to Reduce the Viscosity of a Liquid or Supercritical Fluid Mobile Phase

[0100] The fourth embodiment of this application is the use of microwave radiation to reduce the viscosity of the mobile phase in a Liquid or Supercritical Fluid Separation. The viscosity of a fluid is, in part, a function of the intermolecular forces between the molecules that constitute the fluid. For example, the viscosity of butanol is 2.95 cP and the viscosity of pentane is 0.24 cP, despite the fact that these molecules are very similar in size and shape. This is because the former is more polar than the latter and, therefore, the intermolecular forces between the molecules are stronger. It is known that increasing the temperature of a liquid results in a substantial drop in viscosity, due to the resulting higher kinetic energy of the molecules, which weakens these intermolecular interactions (Teutenberg in High Temperature Liquid Chromatography, RSC Publishing, 2010). It is thought that the application of microwave radiation would have a similar effect, due to the enhanced molecular motion that is caused. In particular, the unique nature of the alignment and randomization process that results from the application of microwave radiation, and the resulting rotation of the fluid molecules, would be expected to disrupt, or weaken, intermolecular interactions, which contribute to the higher viscosities of polar liquids. This should result in a lower viscosity and, consequently, a lower back pressure in chromatographic separations. This effect should work in the absence of any significant increase in temperature; but may be most pronounced if microwave radiation is applied in combination with some increase in temperature.

[0101] A subset of this embodiment would be the application of microwave radiation only during the intervals within a separation where peak-free regions of the chromatogram are being eluted, or perhaps where non-critical peak pairs are being eluted. This would have the effect of reducing the viscosity and allowing higher flow rates to be utilized during these time intervals. During these peak free or non-critical peak times of the separations one may not be concerned with the connection between the flow rate and the efficiency of the separation. Therefore, higher flow rates could be exploited, during these times, to speed the analysis.

Fifth EmbodimentUse of Microwave Radiation to Apply Temperature Pulses to Selected Time Segments of the Separation

[0102] It has already been established that microwave radiation has the potential to heat liquids more rapidly than is possible by conventional heating methods (air ovens, block heaters, etc.). In addition, the use of microwave radiation makes it possible to heat only the mobile phase and analytes but not the column itself (assuming, of course, that the column and stationary phase particles are constructed of material that does not absorb microwave radiation to any significant degree. Thus, for example, columns made with stainless steel housing are probably not the most appropriate for this technique). Therefore, the time required for the system to subsequently cool is much shorter than with conventional heating methods, because once the hot liquid is pumped away most of the heat is gone as well. Generally, all that remains would be a moderately elevated temperature due to heat that was convectively transferred to the column housing and stationary phase particles.

[0103] Use of microwave radiation to accomplish temperature gradients has been presented in the literature (Terol, Maestre, Prats, and Todoli, 2012) and, therefore, is not claimed as part of this application. However, a related approach would be the application of temperature pulses to selected segments of the chromatogram to help optimize resolution. Thus far, this has only been accomplished by conventional heating methods (Causon, Cortes, Shellie, Hilder, 2012).

[0104] However, the use of microwave induced heating is ideally suited to accomplish these rapid temperature pulses as microwave radiation makes possible very fast increases in temperature. And, as mentioned above, faster rates of cooling are possible due to the fact that the microwave heats only the mobile phase and the analytes. It should be mentioned that use of insulating material, or a vacuum, either surrounding the column or as part of the column itself, is advisable with this technique since it would help to prevent radial temperature gradients from developing when heating of the mobile phase occurs. And, in fact, use of insulation in this way could be helpful for any application of microwave radiation to liquid or supercritical fluid chromatography.