Patent Application
20100012584
A subject of the invention is a novel method for enrichment by chromatography using a liquid mobile phase containing dissolved gas as well as its use for enriching a compound of a mixture in general, and the use of known devices for its implementation.
Various chromatography methods are known, using very varied eluents or solvents, promoting the migration of solutes in the column. Gas phase, liquid phase chromatography (also referred to as High Performance Liquid Chromatography or HPLC), in supercritical or subcritical phase, referred to as SFC for Supercritical (or Subcritical) Fluid Chromatography are known. The eluents used can be pure substances or mixtures, by way of example in the field of supercritical or subcritical chromatography, a co-solvent which can be for example an alcohol is generally used in addition to CO2.
The term “supercritical state” denotes a state characterized either by a pressure and a temperature respectively greater than the critical pressure and temperature of the substance in the case of a pure substance, or by a pressure and temperature respectively greater than the critical pressure and temperature of the mixture in the case of a mixture. Supercritical fluids have remarkable properties compared with liquids, in particular a lower viscosity and greater diffusivity, which improves separation by chromatography. Chromatography can also be applied to so-called “non-supercritical” fluids referred to as “subcritical”, i.e. in a state characterized either by a pressure greater than the critical pressure and by a temperature lower than the critical temperature in the case of a pure substance, or by a pressure greater than the critical pressures of each of the components of the mixture in the case of a mixture (on this subject, reference may be made to the article by Michel Perrut in Informations Chimie 321, October 1990, pages 166 to 177, “les fluides supercritiques, applications en abondance”).
Whether the eluent is supercritical or subcritical, the pressures are always very high, requiring equipment, as well as operating conditions, suited to these very high pressures.
J Microcolumn Separations 7(5) 477-483 (1995) describes an analytical chromatography system making use of a methanol/H2O/CO2 eluent 49/21/30 (mol) at 204 atm. At 26° C., the pressure drop at the outlet of 4 columns is 144.8 atm, whereas for 60° C., the pressure drop is 81.9 atm. Thus, the outlet pressures for the methanol/H2O/CO2 mixture are established at 59.2 atm and 122.1 atm for 26 and 60° C. respectively. In the case of use at 26° C., the pressure at the outlet of the columns is lower than the critical pressure, whereas it is greater than the critical pressure for the higher temperature in comparison with the critical pressure of the CO2 alone. This article defines the “Enhanced-Fluidity Liquid mobile phase” eluent used in “Enhanced-Fluidity Liquid Chromatography” (EFLC), as being a standard HPLC eluent to which significant proportions of a low-viscosity fluid such as CO2 have been added.
Anal. Chem 1998, 70, 3298-3303 describes an analytical chromatography system using an EFLC type eluent, in this case THF/CO2 at 260 atm (60/40 and 70/30). The pressure drop values shown in this article were measured on a 2 m capillary column and are difficult to compare with standard 25 cm columns and sometimes (in the case of high flow rates) exceed the pressure in the eluent pump (3000 psi).
Anal. Chem 1999, 71, 2139-2145 describes an analytical chromatography system using various eluents (ethanol/hexane/CO2, methanol/CO2, at 170 atm which are of the EFLC type, this document indicating that EFLC type chromatography involves the use of liquefied gases such as CO2 and CHF3, combined with polar liquids such as ethanol, as mobile phase. It is indicated that the pressure drop recorded with EFLC type solvents is smaller, which increases the speed of analysis. The outlet pressures are however still very high in this document, the inlet pressure being fixed at 170 atm, whereas the pressure drop is approximately 21 bar at most (for 10% of CO2 added). The outlet pressure is in this case still higher than the critical pressure (in fact 170 bar minus 21 bar pressure drop is a value clearly greater than the pressure of approximately 74 bar, the critical pressure of CO2 alone).
Chirality 9:672-677 (1997) describes a chromatography separation method under different operating conditions for the solvent. In particular, this article studies the effects of the addition of CO2 on the critical points (pressure and temperature). The article studies the methanol/CO2 binary system, for variable quantities of CO2. The binary system studied is the methanol/CO2 eluent with 10 mol. % of CO2. For this binary system, the critical pressure and temperature are 114 atm and 60° C., respectively. The article studies the effect of the operating conditions on the selectivity, retention and resolution. The article concludes that the optimum resolution is obtained with a supercritical pressure and a subcritical temperature.
The four articles above deal exclusively with analytical chromatography.
A need still remains for chromatography separation methods which are effective, which do not require operating conditions which are too restrictive, but which especially make it possible to collect a mass of enriched or purified product in quantities as large as possible.
None of the above documents describes or suggests the present invention.
The invention therefore provides a method of enriching by chromatography one or more compounds of a mixture in at least one column using an eluent, characterized in that a liquid mobile phase containing dissolved gas is used as eluent.
According to an embodiment, the outlet pressure is lower than the critical pressure of the eluent gas.
According to an embodiment, the method is implemented on at least one column having a length-to-diameter L/D ratio of less than 10, for example less than 5.
According to an embodiment, the average linear velocity during the implementation of the enrichment by chromatography is greater than 0.3 cm/s.
According to an embodiment, the injected volume of the solution containing said compound to be enriched represents between 1 and 20% of the void volume of said at least one column, preferably between 2 and 10%.
According to an embodiment, the injection of said mixture into said at least one column is carried out with a liquid.
According to an embodiment of the invention, the eluent is at a temperature lower than the critical temperature of the eluent gas.
According to an embodiment of the invention, the eluent is at a supercritical temperature and at a column outlet pressure lower than the critical pressure of the eluent gas.
According to an embodiment of the invention, the eluent according to the invention is constituted by a liquid mobile phase, said liquid mobile phase contains dissolved gas, preferably dissolved carbon dioxide, and surprisingly, remains homogeneous under the temperature and pressure conditions as defined according to the present invention.
According to an embodiment of the invention, the eluent is at a pressure which corresponds to the minimum pressure at which the eluent would be a homogeneous phase, increased by a value comprised between 1 and 20 bar, preferably between 3 and 10 bar at the outlet of said at least one column.
According to an embodiment of the invention, the eluent comprises a liquid mobile phase containing a gas chosen from carbon dioxide, nitrogen protoxide, light alkanes and fluorinated gases.
According to an embodiment of the invention, the eluent comprises a liquid mobile phase containing dissolved carbon dioxide.
According to an embodiment, the eluent according to the invention is constituted by a homogeneous liquid mobile phase containing dissolved gas at a subcritical pressure and at a supercritical temperature.
According to an embodiment, the eluent according to the invention is constituted by a homogeneous liquid mobile phase containing dissolved gas at a subcritical pressure and at a subcritical temperature.
According to an embodiment of the invention, the eluent liquid is chosen from water in a mixture with the miscible organic solvents, the primary alcohols containing 1 to 4 carbon atoms, the alkanes containing 5 to 10 carbon atoms, acetone, acetonitrile, cyclic ethers and esters.
According to an embodiment of the invention, the eluent is binary, ternary or a mixture with 4 or more components.
According to an embodiment of the invention, the gas content in the eluent is comprised between 5 and 95% by volume, preferably between 10 and 50% by volume.
According to an embodiment of the invention, the method is implemented on a silica-based stationary phase or also implemented on a chiral stationary phase.
According to an embodiment of the invention, the gas is dissolved in the liquid eluent by maintaining sufficient pressure to obtain a homogeneous liquid phase throughout the length of the chromatographic column.
According to an embodiment, the invention relates to the use of the eluent according to the invention, in any preparative chromatography device, continuous or in sequence with at least one column, for enriching one or more compounds of a mixture.
According to an embodiment, the invention relates to the use of an eluent constituted by a liquid mobile phase containing dissolved gas in chromatography systems comprising axial compression columns.
The invention also relates to the use of a preparative chromatography device for implementing the method according to the invention.
According to another embodiment, the invention relates to the use in preparative chromatography of an eluent composed of a fluid mobile phase containing a dissolved gas and at an outlet pressure lower than the critical pressure of the eluent gas and at a temperature lower than the critical temperature of the eluent gas.
According to another embodiment, the invention relates to the use in preparative chromatography of an eluent which is a single phase mixture of a gas and a liquid at a pressure which corresponds to the minimum pressure at which the eluent would be a homogeneous phase, increased by a value comprised between 1 and 20 bar, preferably between 3 and 10 bar.
A subject of the invention is also the use of a subcritical or supercritical preparative chromatography device for the implementation of the method according to the invention.
A subject of the invention is also the use in preparative chromatography of an eluent composed of a fluid mobile phase containing a dissolved gas and at an outlet pressure lower than the critical pressure of the eluent gas and at a temperature lower than the critical temperature of the eluent gas.
A subject of the invention is also the use in preparative chromatography of an eluent which is a single phase mixture of a gas and a liquid at a pressure which corresponds to the minimum pressure at which the eluent would be a homogeneous phase, increased by a value comprised between 1 and 20 bar, preferably between 3 and 10 bar.
The invention offers numerous advantages compared with a standard SFC system. In particular, the operating pressure is lower and/or the solvency is higher. It is possible to carry out the separation with the eluents according to the invention under the pressure conditions conventionally used in HPLC. The installation using the eluents according to the invention, compared with a standard SFC separation device, does not require a high-pressure CO2 pump, a CO2 condenser, a cooling bath, a CO2 evaporator, a hot bath or finally, high-pressure separators. However, a person skilled in the art will appreciate that the SFC equipment is compatible with the eluents according to the invention
The invention makes it possible, compared with HPLC type chromatography, and for a comparable linear velocity, to increase the number of plates and reduce the pressure drops, which leads to better system performances.
The invention can therefore be implemented on standard systems, whether these are SFC or HPLC.
a and 7b represent the separation chromatograms in an elution mode according to the state of the art and according to the invention, in the case of an overloaded injection.
The invention is implemented with all the chromatography techniques using a stationary phase and an eluent in the state according to the invention.
The chromatography according to the invention can be continuous, discontinuous (batch) or sequential (in particular known as SMB). The invention can be implemented on a single column, or in a multi-column device, single-column devices being preferred here.
SMB technology has been known for a long time, and is the subject in particular of the following patents: U.S. Pat. No. 2,957,927, U.S. Pat. No. 2,985,589, U.S. Pat. No. 3,205,166, U.S. Pat. No. 3,291,726 and U.S. Pat. No. 3,310,486 (UOP). Columns with variable chromatographic lengths can also be used in the invention. Thus, it is possible to implement the invention in a so-called Varicol® system, developed by the Applicant, and corresponding to the patents U.S. Pat. No. 6,136,198, U.S. Pat. No. 6,375,839, U.S. Pat. No. 6,712,973, U.S. Pat. No. 6,413,419 and WO 00/25885. It is also possible to implement the invention in a so-called Cyclojet® system, and corresponding to the patents U.S. Pat. No. 5,630,943 and WO 97/20206, as well as U.S. Pat. No. 6,063,284 and WO 98/51391.
Any other chromatographic method, including batch chromatography, whether or not multi-column, can be used; there can be mentioned the systems known under the names of ModiCon® and PowerFeed®, as well as two-zone SMB chromatography.
The use of supercritical solvents in a multi-column device is described in particular in WO93/22022.
The invention uses the standard phases, such as for example silica-based stationary phases, reversed-phase adsorbents or also chiral stationary phases (CSP). The CSP phase which can be used in the invention can be (i) a cellulose derivative (e.g. esters or carbamates, preferably deposited on silica), optionally grafted, (ii) tartrate phase, (iii) acid and basic CSP phase (Pirkle phase), (iv) an amylose derivative (e.g. esters or carbamates. preferably deposited on silica, optionally grafted), (v) polyacrylamide phase and (vi) others.
Any phase can be used and is determined in standard fashion by routine tests by a person skilled in the art. A suitable stationary phase will exhibit a balance of the following properties a) retention time; b) selectivity; c) load capacity; d) productivity; and e) eluent consumption.
All types of column are suitable for the implementation of the invention. However, it is preferable to use preparative chromatography columns, having a sufficient diameter. Columns having length to diameter L/D ratios less than 10, advantageously less than 5 are preferably used.
With reference to
The eluent used in the invention is a specific eluent. The eluent according to the invention is a mixture of a gas and a liquid, in a homogeneous phase (or single phase). The terms gas and liquid correspond to the states of the pure substances at an ambient temperature of 23° C. and under normal pressure. For example, CO2 is classified as a gas in the invention, whereas methanol or ethanol is classified as a liquid in the invention. The eluent according to the invention is not supercritical or subcritical, as it is at a pressure lower than the critical pressure (whereas the supercritical or subcritical eluents are at a pressure which is greater than the critical pressure of the fluid considered). The eluent in the invention is at a pressure lower than the critical pressure, for example at least 5 bar lower than this critical pressure. The pressure of eluent according to the invention is an operating parameter which ensures the homogeneity of the mobile phase. The gas used in the mobile phase is thus ‘dissolved’ in the liquid or liquids used in order to produce the eluent according to the invention. Examples of gases capable of being used in the invention are: carbon dioxide, nitrogen protoxide, the light alkanes (in particular containing 1 to 4 carbon atoms, such as ethane, ethylene), and fluorinated gases (CHF3).
Examples of liquids capable of being used in the invention are: water in a mixture with the miscible organic solvents (for example water and alcohol or water and acetonitrile, the quantity of water being preferably less than 50% by volume), primary C1-C4 alcohols (for example methanol, ethanol, isopropanol), alkanes (in particular containing 5 to 10 carbon atoms, such as hexane, heptane), acetone, acetonitrile, cyclic ethers (for example THF), esters (for example ethyl acetate) and all the organic solvents used for liquid and reversed mobile phase chromatography.
It is possible to form binaries, ternaries, or mixtures with 4 or more components. In general, a gas and a liquid or a gas and two liquids are used.
The quantity of gas dissolved in the liquid is variable, and can be comprised between 5 and 95%, preferably between 10 and 50% by volume. (The use of quantities by volume is practical, because most chromatographic systems use volumetric pumps). The optimum gas content is determined by experimentation.
The eluent according to the invention is “inflated” with the dissolved gas, followed by a reduction in the viscosity and increase in the diffusivity, these two parameters being significant in the chromatographic application.
It is also noted that the solvency of the eluents according to the invention cannot be modified by the addition of gas to the liquid. But it is also possible, by adjusting the pressure and the quantity of gas and more preferably in the case of CO2, to modify the solvency. Reference can be made, by way of example, to the document Supercrit. Fluids, vol 23, 2002. p 195, for an illustration of the solubility of salicylic acid in propanol as a function of the temperature and pressure of CO2.
To the extent that it is possible to adjust the properties of the eluent (viscosity, diffusivity, solvency), it is possible to qualify this eluent as neoteric (it is a “tunable” solvent).
The present method is an enrichment method; unlike the methods of the state of the art using mixtures of liquid and gas, the method according to the invention leads to isolated fractions which are used. The invention is the use in preparative chromatography of an eluent which is a liquid mobile phase containing a dissolved gas.
The operating conditions used in the invention are defined in relation to the quantity of gas in the eluent, the type of gas, the type of liquid, and the temperature. It is possible to define an operating pressure according to these elements. In general, the outlet pressure of the chromatography device is fixed and the flow rate is then adjusted (which leads to a given pressure drop).
By way of example, for a standard binary of the methanol/CO2 type, the operation is carried out below the critical pressure, therefore as a first approximation towards 100-110 bar at maximum. The operation is also carried out above the pressure of the appearance of the first bubbles of gas, i.e. as a first approximation towards 30 bar. The zone corresponds to zone 3 in
With reference to
The operating conditions can be defined more precisely, in particular by determining the minimum pressure at which the chromatography is implemented such that the eluent remains in homogeneous phase in all of the chromatographic zones.
It is possible to use for example the calculated phase envelope method. A phase envelope is calculated for a given binary. This method is described in Pascale Borg, Jean-Noel Jaubert and Felicie Denet, (Fluid Phase Equilibria Vol 191, Issues 1-2, 2001, pp. 59-69).
It is also possible to use the data which are published in the literature, for certain mixtures. For example, for the ethanol/CO2 mixture, it is possible to find these experimental values.
Once the minimum pressure has been determined, for reasons of reliability, the operating pressure at the column outlet is generally fixed at a slightly higher value, for example higher than 5 bar. The operating pressure is therefore Pmin+5. Typically, the pressure Pmin is determined and between 1 and 20 bar, preferably between 3 and 10 bar is added.
The invention can be implemented under specific linear velocity and injected volume conditions.
As regards the linear velocity, it is significantly higher than in the state of the art indicated in the introductory part. In the case of HPLC, the linear velocity is typically of the order of 0.1-0.2 cm/s. In the case of the state of the art relating to EFLC, the linear velocity is of the order of 0.2 cm/s. By way of comparison, the linear velocity, in the invention, is typically greater than 0.3 cm/s.
Such a linear velocity has the advantage that the products exit more rapidly. There is therefore a gain in cycle time: the injection is carried out more often, the production is increased. The drawback conventionally associated with a high linear velocity is that the pressure increases in the column (as the fluid passes through a porous medium). But as the eluent according to the invention has a lower viscosity, the pressure drop is reduced, thus the conventional drawback is compensated for by the quality of the eluent according to the invention.
Another drawback conventionally associated with a high linear velocity is that increasing the velocity involves the risk of reducing the efficiency of the columns. In fact, if the velocity in the columns is increased, this generally promotes broadening of the peaks. Moreover, in the preparative application, the quantity of mixture to be separated (“overloaded injection”) is increased, in order to saturate the phase. Thus, increasing the velocity in the case of a high load, or even of an overloaded injection, is not desirable. Surprisingly, the Applicant has demonstrated that the risk of reducing the efficiency of the column has not proved sufficiently significant to lower the quantity of injectable product allowing a good separation of the products.
The quantity injected in a preparative separation method is significantly greater than in an analytic method, generally the behaviour of the column is no longer linear in a preparative separation method.
The volume of the solution containing the separated products injected into the column can moreover be relatively significant. In the case of the analytical application, the injected volume, as a % of the volume of the empty column, is generally less than 1%. In the case of the invention, the Applicant has surprisingly noted that the injected volume could be increased by as much as several percent, typically up to 20%, without posing any problem. Whilst it can easily be understood that a small injected volume (<1%) does not disturb the system, this is not the case with much larger volumes. The phenomenon is still more surprising when the injection is carried out not with the eluent of the invention, but with only the liquid phase of this eluent.
The invention can nevertheless also use injection loops known in chromatography systems. The mixture to be separated is injected into the loop, then it penetrates into the column. This system is in particular used in SFC. It is possible in the case of the invention to put such a loop in place, with the same eluent in the loop as in the columns. But it is preferable, being simpler, to use only the eluent liquid as injection vehicle.
Thus, the invention provides a method for separating or enriching by chromatography a mixture in at least one column using an eluent, said at least one column being a preparative chromatography column (in particular having an L/D ratio less than 10), the eluent used being a liquid mobile phase containing dissolved gas.
The method according to the invention allows implementation at an average linear velocity greater than 0.3 cm/s and with an injected volume of mixture to be separated which represents between 2 and 20% of the void volume of the chromatography column or columns.
The method according to the invention allows implementation with an injection of said mixture into the column with a liquid (that of the eluent) and with an injected volume of mixture to be separated which represents between 1 and 20% of the void volume of the chromatography column.
The invention is moreover implemented on conventional chromatographic beds. These beds can also be compressed into axial compression columns or into columns for packing and compression of the Pack-n-Sep® type rapid beds (Novasep). The analytical columns are filled with the phase according to a general flash method. In the case of columns with large diameters, i.e. preparative columns, the column is loaded with the phase which is generally in the same solvent as that used for the chromatography. This principle is not applicable to the eluent according to the invention, since it must be handled under pressure. In fact, the phase can be loaded and optionally compressed in the liquid solvent included in the composition of the eluent according to the invention. This use of liquid which is not the same as that used during the chromatography surprisingly poses no operating problem.
The eluent, once it has entrained the solute, becomes isolated fractions. These fractions are treated in a standard fashion in order to separate the dissolved compounds. In general, the gas is lost but the liquid can be recycled. A recycling of gas can nevertheless be provided, if appropriate.
The following examples illustrate the invention without limiting it.
The TSO (trans-stilbene oxide) enantiomers are separated under the following conditions, corresponding to the conditions of analytical chromatography.
Chiralcel Column OD-20 μm, 4.6 mm×25 cm (Chiral Technologies Europe, Illkirch, France)
Temperature 20° C.
Outlet pressure 1 bar (in HPLC mode) and 50 bar in the mode according to the invention with the eluent according to the invention
Liquid eluent: heptane/IPA 70/30 (v/v)
Flow rate of non-expanded eluent=1.4 ml/mm
CO2 expansion rate 30 and 40%
Pressure drops 9 and 10 bar, respectively.
Volume/quantity of TSO injected: 20 μl/0.64 mg
The TSO (trans-stilbene oxide) enantiomers are separated under the following conditions, corresponding to the conditions of the preparative chromatography, the injection this time being “overloaded”, which corresponds to the normal conditions of use of preparative chromatography.
Chiralcel Column OD-20 μm, 4.6 mm×25 cm (Chiral Technologies Europe, Illkirch, France)
Temperature 20° C.
Outlet pressure 1 bar (non-expanded solvent) and 43 bar with the eluent according to the invention
Liquid eluent: heptane/IPA 70/30 (v/v)
Flow rate of non-expanded eluent=1 ml/min
Flow-rate of expanded eluent=2 ml/min
CO2 expansion rate 30%
Pressure drops of 12 and 11 bar, respectively
Volume/quantity of TSO injected: 100 μl/3.2 mg, 200 μL/6.4 mg, 250 μl/8 mg;
Injection solvent: isopropanol (IPA)
The overload chromatogram (
The linear velocity in the mode according to the invention is of the order of 0.334 cm/s (under the same conditions as in Example 1).
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/FR06/01997 | Aug 2006 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR07/01395 | 8/23/2007 | WO | 00 | 2/26/2009 |
