Use of compressed gas for moving eluent applied to chromatography

Abstract

The invention relates to a method for chromatographic separation, comprising at least one step of elution of species held on a stationary phase by means of an eluent, followed by a step of moving the eluent in contact with the stationary phase by means of a compressed gas. Preferably, the movement step takes place after a step of elution of the product(s) of interest and/or after a step of regeneration of the stationary phase.

Claims

1. A method for chromatographic separation, comprising at least one step of eluting species retained on a stationary phase by means of an eluent, followed by a step of displacing the eluent in contact with the stationary phase by means of a compressed gas, wherein the compressed gas is in a liquid state or a supercritical state.

2. The method of claim 1, wherein the eluent is a single compound.

3. The method of claim 1, wherein the eluent is a mixture of at least two compounds.

4. The method of claim 1, wherein the eluent is an aqueous and/or organic solvent, or a mixture of aqueous and/or organic solvents.

5. The method of claim 1, wherein the eluent is a mixture of aqueous and/or organic solvent(s) and compressed gas.

6. The method of claim 1, comprising, after the step of displacing the eluent, collection of a mixture of compressed gas and aqueous and/or organic solvent(s) at the output of the stationary phase, and separation of the compressed gas and the aqueous and/or organic solvent(s).

7. The method of claim 6, wherein the aqueous and/or organic solvent(s) are recycled.

8. The method of claim 6, wherein the compressed gas is recycled.

9. The method of claim 1, wherein the step of eluting species retained on a stationary phase by means of an eluent is an elution step of product(s) of interest.

10. The method of claim 1, wherein the step of eluting species retained on a stationary phase by means of an eluent is a step of regeneration of the stationary phase.

11. The method of claim 1, wherein the eluent is a first eluent, and the method comprises, after the step of displacing the eluent by means of a compressed gas, bringing into contact a second eluent with the stationary phase.

12. The method claim 11, wherein bringing into contact a second eluent with the stationary phase is a step of regeneration of the stationary phase.

13. The method claim 11, wherein bringing into contact a second eluent with the stationary phase is an equilibration step.

14. The method of claim 1, which comprises, cyclically: an equilibration step, comprising bringing an equilibration eluent into contact with the stationary phase; an injection step comprising bringing a composition comprising a product to be separated into contact with the stationary phase; an optional rinsing step, comprising bringing a rinsing eluent into contact with the stationary phase; a step of eluting the product, comprising bringing an eluting eluent into contact with the stationary phase and collecting the product to be separated; again, the equilibration step; and wherein the step of displacing eluent in contact with the stationary phase by means of a compressed gas is performed between the product elution step and the equilibration step.

15. The method of claim 1, which comprises, successively or cyclically: an equilibration step, comprising bringing an equilibration eluent into contact with the stationary phase; an injection step comprising bringing a composition comprising a product to be separated into contact with the stationary phase; an optional rinsing step, comprising bringing a rinsing eluent into contact with the stationary phase; a step of eluting the product, comprising bringing an eluting eluent into contact with the stationary phase and collecting the product to be separated; a regeneration step, comprising bringing a regeneration eluent into contact with the stationary phase; if applicable, the equilibration step again; wherein the method comprises at least one displacement step of an eluent in contact with the stationary phase by means of a compressed gas, between the product elution step and the regeneration step; and/or between the regeneration step and the equilibration step.

16. The method of claim 15, which comprises both a displacement step of an eluent in contact with the stationary phase by means of a compressed gas between the product elution step and the regeneration step, and another displacement step of an eluent in contact with the stationary phase by means of a compressed gas between the regeneration step and the equilibration step.

17. The method of claim 14, wherein the steps of equilibration, injection, optional rinsing, elution, and optional regeneration, are implemented with eluents comprising a compressed gas.

18. The method of claim 14, wherein the steps of equilibration, injection, optional rinsing, elution, and optional regeneration, are implemented with eluents not comprising a compressed gas.

19. The method of claim 1, wherein the stationary phase comprises at least one material selected from the group consisting of activated alumina, silica gel, activated carbon, silicic acid, magnesium oxide, calcium hydroxide, magnesium hydroxide, cellulose, amylose, a polymerized adsorbent, a derivative thereof, and combinations thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the drawbacks of the prior art, by showing the evolution of the concentration of different solvents at the outlet of a chromatography column of 50 mm internal diameter, filled with a non-grafted silica stationary phase. The elution time in minutes is shown on the abscissa, and the solvent content in % is shown on the ordinate. Initially, the column filled with hexane (solvent 1). At t=10 minutes, the injection of acetone at 100% by weight (solvent 2) is initiated. At t=15 minutes, the injection of dichloromethane at 100% by weight (solvent 3) is initiated. At t=25 minutes, the injection of hexane at 100% by weight is again initiated (solvent 1).

(2) FIG. 2 very schematically shows a succession of steps in a method according to the invention.

(3) FIG. 3 very schematically shows a succession of steps in a preferred embodiment of the invention (with a compressed gas content in the output stream of the chromatographic column on the ordinate, and a duration on the abscissa).

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

(4) The invention is now described in more detail and without limitation in the description which follows.

(5) In its most general form, the invention relates to a chromatographic separation method, wherein a step of eluting species retained on a stationary phase by means of an eluent is followed by a step of displacing the eluent in contact with the stationary phase by means of a compressed gas.

(6) By “eluent” is meant a fluid capable of desorbing species retained on a stationary phase, whether they are species of interest or impurities.

(7) An eluent may be a pure solvent or a mixture of solvents. Each solvent may be, notably, aqueous or organic, or even a gas in the liquid or supercritical state or close to the supercritical state.

(8) Organic compounds which may be used as solvents are, in particular, alkanes, alcohols, ethers, esters, ketones and nitriles.

(9) The alkanes are preferably C1-C10, more preferably C6-C8. They may be linear or branched, and preferably linear. Hexane and heptane are preferred examples.

(10) The alcohols are preferably of the formula R—OH where R is a C1-C6 alkyl group. They include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol and t-butanol.

(11) The ethers are preferably of the formula R—O—R′, where R and R′ are C1-C6 alkyl groups. They include diethyl ether, diisopropyl ether, and methyl t-butyl ether (MTBE).

(12) The ethers are preferably of formula R—(C═O)O—R′, where R and R′ are C1-C6 alkyl groups. They include methyl acetate and ethyl acetate.

(13) The ketones are preferably of formula R—(C═O)—R′, where R and R′ are C1-C6 alkyl groups. They include acetone, methyl ethyl ketone and methyl isobutyl ketone (MIBK).

(14) The nitriles are preferably of formula R—CN, where R is a C1-C6 alkyl group. They include acetonitrile.

(15) By “compressed gas” is meant a fluid which is in the gaseous state under normal conditions of temperature and pressure (i.e. 0° C. and 1 bar absolute), but which is used in the method of the invention at a pressure above the pressure of 1 bar absolute.

(16) According to particular embodiments, the compressed gas is used at a pressure greater than or equal to 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 bar absolute.

(17) In order to simplify the implementation of the method, it may be advantageous that the pressure of the compressed gas used for the displacement step is less than or equal to 300 bar, preferably 200 bar, preferably 150 bar, preferably 100 bar, preferably 75 bar, and more preferably 50 absolute.

(18) The compressed gas may be in the gaseous state when used in the method of the invention for carrying out the eluent displacement step. However, preferably, it is in the liquid or supercritical state.

(19) As examples of compressed gases, mention may be made of carbon dioxide (CO.sub.2), propane and nitrous oxide. CO.sub.2 is preferred. It has the particular advantage of presenting a critical point at near-ambient temperature (31° C.) and at moderate pressure (73 bar). It is also already widely used as an eluent in chromatography in liquid or supercritical phase, pure or mixed with other solvents.

(20) The chromatographic separation according to the invention may be a liquid chromatographic separation, in which case the eluents used are liquid solvents as described above, preferably aqueous and/or organic.

(21) Alternatively, it may be a chromatography with liquid expanded by a gas, in which case the eluents used are mixtures of solvent or liquid solvents (preferably organic) as described above, and compressed gas, wherein the liquid solvent(s) are predominant by weight.

(22) Alternatively, it may be a supercritical chromatography, in which case the eluents used are either one or more compressed gas(es) alone, or one or more mixtures of compressed gas and one or more liquid solvents (preferably organic) wherein the compressed gas is predominant by weight.

(23) However, it should be understood that the compressed gas used during the eluent displacement step is different from the eluent itself, even when this eluent is also a compressed gas.

(24) The method of the invention may comprise, notably, successively or cyclically: an equilibration step; a step of injecting a composition containing at least one product to be separated (or purified); an optional rinsing step; a step of eluting the product; an optional regeneration step; again the equilibration step if the method is implemented cyclically.

(25) One or more eluents, as defined above, are used at each of these steps and brought into contact with the stationary phase.

(26) According to one embodiment, the equilibration eluent and the eluting eluent are identical.

(27) According to one embodiment, the rinsing eluent and the eluting eluent are identical.

(28) According to one embodiment, the equilibration eluent and the rinsing eluent are identical.

(29) The optional rinsing step intends to desorb from the stationary phase species having an affinity for the stationary phase lower than that of the product to be separated.

(30) The optional regeneration step intends to desorb from the stationary phase species having an affinity for the stationary phase higher than that of the product to be separated.

(31) Preferably, the regeneration eluent is different from the eluting eluent. Advantageously, it has a higher affinity for the stationary phase than the eluting eluent.

(32) At each step where an eluent is used, an eluent of constant composition may be used (elution in isocratic mode, wherein the eluting force remains constant over time).

(33) Alternatively, it is possible to use an eluent of composition varying over time, for example two or three successive different eluents, or an eluent having a composition that varies continuously (elution in gradient mode, wherein the eluting force then generally increases over time). To do this, it is, for example, possible to gradually increase the proportion of the most eluent solvent in a mixture containing at least two solvents.

(34) In the context of the succession of steps described above, the method of the invention comprises at least one step of displacement of eluent by a compressed gas.

(35) This displacement step may be performed, notably, after the elution step and before the regeneration step (displacement of the eluting eluent).

(36) It may also be performed after the regeneration step and before the equilibration step (displacement of the regeneration eluent).

(37) When the method does not comprise a regeneration step, the displacement step may be carried out after the elution step and before the equilibration step (displacement of the eluting eluent).

(38) In a preferred embodiment, a regeneration step is present, and two displacement steps are provided: one after elution and before regeneration (displacement of the eluting eluent); and the other after regeneration and before equilibration (displacement of the regeneration eluent).

(39) This embodiment is illustrated in FIG. 2, which shows schematically the displacement of eluents in the case where the compressed gas is CO.sub.2: two cycles (1 and 2) are represented. Each cycle comprises successively: an equilibration step (Eq.), a product injection step (I), a step of eluting the slightly retained impurities and the product (EI.), An eluent displacement step by CO.sub.2, a regeneration step, namely desorption of highly retained impurities (R), and finally another displacement step of the solvents by CO.sub.2.

(40) According to one embodiment, all the steps of the method are carried out substantially at the same pressure. According to an alternative embodiment, the pressure differs from one step to another. Notably, it may be higher in the eluent displacement steps by means of the compressed gas.

(41) At the end of each eluent displacement step, preferably at least 95% by weight, more preferably at least 99% by weight, or at least 99.5% by weight, or at least 99.9% by weight, or at least 99.95% by weight, or at least 99.99% by weight of the eluent initially in contact with the stationary phase, is removed.

(42) During the eluent displacement step, an output stream is collected at the output of the stationary phase. Preferably, at the end of the displacement step, the content of eluent in the outlet stream is less than or equal to 5% by weight, more preferably less than or equal to 0.1% by weight, or less than or equal to at 0.5% by weight, or less than or equal to 0.1% by weight, or less than or equal to 0.05% by weight, or less than or equal to 0.01% by weight.

(43) The stationary phase is generally present as a bed in an enclosure, which, in general, is in the form of a column.

(44) The chromatographic separation according to the invention may thus be implemented either in a single-column chromatography system or in a multi-column chromatography system.

(45) It may be a chromatography system with static bed or not. In a static bed chromatography system, the mixture of compounds to be separated percolates in an enclosure or column, which is generally cylindrical. The column contains a bed of porous material (the stationary phase) that is permeable to fluids. The rate of percolation of each compound in the mixture depends on the physical properties of the compound. The compounds most retained on the stationary phase percolate more slowly than the compounds least retained on the stationary phase. This principle makes it possible to perform the desired separation.

(46) It is possible to perform such treatment in several columns in series or in parallel, but generally a chromatographic separation in a static bed system is implemented with a single column.

(47) Examples of such static bed chromatography systems are HPLC (High Performance Liquid Chromatography) or CYCLOJET™ systems (i.e. stationary recycling system).

(48) The CYCLOJET™ system is as described in document U.S. Pat. No. 6,063,284, to which reference is expressly made. It is a discontinuous chromatography separation system with a single column, in which the (i) most retained and then the (ii) least retained species are collected separately at the outlet of the column, wherein a non-separated portion of the chromatogram is recycled by a main pump. The mixture to be separated is periodically injected into the recycled portion of the chromatogram by means of an injection loop. The injection loop is preferably connected between the main pump and the column. After several chromatographic cycles, the method reaches a periodic steady state in which the quantity of products injected is equal to the quantity of products collected separately at the outlet of the column.

(49) According to one embodiment, the chromatographic separation in a single-column static bed system with stationary recycling, is cyclic and comprises the following steps: establishment and maintenance of a chromatographic profile circulating in the column by means of an eluent pump; injection into said circulating chromatographic profile of a sample comprising the at least two compounds to be separated, in a discontinuous manner and at each cycle, the injection being carried out in an injection position by an injection valve by means of a controlled injection loop, in order to inject the sample present in the loop into the circulating chromatographic profile, while the injection valve remains in the injection position from the beginning of the injection until the moment when the entire profile is eluted from the column, then tilting the injection valve to a charging position in order to charge the injection loop when the entire profile is in the column, and collection of at least two fractions enriched from the circulating profile, in a discontinuous and periodic manner.

(50) This separation may also include the following step: passage of eluent in the column as a mobile phase, substantially continuously during the cycle, by means of the eluent pump.

(51) This separation may also include the following steps: recording events occurring from the beginning of the collection of a first fraction to the next beginning of the collection of the first fraction; interruption of the eluent pump during the collection of a third fraction, wherein this interruption continues until the end of the cycle, so that the cycles are temporally reproducible.

(52) According to one embodiment, there is no loss of circulating profile during injection in the maintained circulating profile.

(53) A detailed embodiment of this system may be found in col. 5 I.36 to col. 10 I.41 of document U.S. Pat. No. 6,063,284 cited above.

(54) The chromatographic separation unit may also be a non-static bed chromatography system. A non-static bed system is a multi-column system in which the relative positions of the stationary phase bed and injection and/or flow collection points are displaced over time.

(55) Examples of such non-static bed chromatography systems are SMB, iSMB, SSMB, AMB, VARICOL™, MODICON™, POWERFEED™, DCC or MCSGP.

(56) An SMB system comprises a plurality of individual adsorbent-containing columns, which are connected in series. An eluent stream flows through the columns in a first direction. The injection points of the feed stream and the eluent, as well as the collection points of the separated compounds, are periodically and simultaneously offset by means of a set of valves. The overall effect is to simulate the operation of a single column containing a moving bed of solid adsorbent, wherein the solid adsorbent moves in a countercurrent direction to the eluent stream. Thus, an SMB system is composed of columns that contain stationary beds of solid adsorbent through which the eluent passes, but the operation is such that a continuous countercurrent moving bed is simulated.

(57) The most conventional form of an SMB system is the four-zone SMB system. Other possible forms are three-zone SMB systems and two-zone SMB systems (as described in Kwangnam Lee's article “Two Section Simulated Moving Bed Process” in Separation Science and Technology 35(4):519-534, 2000, which is expressly referred to).

(58) An iSMB system is as described in documents EP 0342629 and U.S. Pat. No. 5,064,539, to which reference is expressly made. An SSMB system cuts the introductions and collection of flows in sub-sequences applied in periodic ways. In the iSMB and SSMB systems, there is at least one step in which the system operates in a closed loop, without product input or output.

(59) Other variants of the SMB systems are: the time-varying SMB system and the POWERFEED™ system, as described in document U.S. Pat. No. 5,102,553 and in the article “PowerFeed operation of simulated moving bed units: changing flow-rates during the switching interval”, by Zhang et al. in Journal of Chromatography A, 1006:87-99, 2003, which is expressly referred to; the MODICON™ system, as described in document U.S. Pat. No. 7,479,228, to which reference is expressly made; and the SMB system with internal recirculation, as described in document U.S. Pat. No. 8,282,831, to which reference is expressly made.

(60) A DCC chromatography system is as described in document FR 2889077, to which reference is expressly made. A DCC system is a sequential method with periodic displacement of the injection points of mobile phase and of mixtures to be separated, having the characteristic of being constantly open loop. It uses two or more columns.

(61) An AMB system has a similar operation to an SMB system. However, instead of displacing injection points of the feed stream and eluent, as well as collection points, by means of a valve system, a set of adsorption units (columns) are physically displaced relative to the feeding and collection points. Again, the operation simulates a continuous countercurrent moving bed.

(62) A VARICOL™ chromatography system is as described in documents U.S. Pat. Nos. 6,136,198, 6,375,839, 6,413,419 and 6,712,973, to which reference is expressly made. A VARICOL™ system comprises a plurality of individual adsorbent-containing columns that are connected in series. An eluent is passed through the columns in a first direction. Unlike the SMB system, the injection points for the mixture to be separated and for the eluent and the collection points of the separated compounds in the system are displaced periodically but asynchronously, by means of a set of valves. The overall effect is to create separation zones of variable length over time, thus dynamically allocating the stationary phase in the zones where it is most useful, and allowing similar separation power with fewer chromatography separation units and increased productivity. Unlike an SMB system, a VARICOL™ system does not simulate the operation of a single column containing a solid adsorbent moving bed, wherein the solid adsorbent moves in a countercurrent direction to the eluent stream, and so the operating principle of VARICOL™ cannot be implemented in an equivalent AMB system.

(63) All of the above known installations may be adapted for implementing the method according to the invention, by providing adequate supplies for the enclosure(s) containing the stationary phase bed, namely: at least one eluent supply as described above, preferably several eluent supplies (notably an eluting eluent supply and a regeneration eluent supply); and at least one compressed gas supply, for the step(s) of eluent displacement by the compressed gas.

(64) The installation of the invention also advantageously comprises a device for separating compressed gas and solvent at the outlet of the enclosure(s) or column(s).

(65) This separation device may, notably, comprise a cyclonic separator, a filter and/or an evaporator.

(66) Thus, the method of the invention advantageously comprises a collection of a mixture of compressed gas and one or more solvents during the step of displacement of the eluent by the compressed gas, and the separation of the compressed gas on the one hand and solvent(s) on the other hand. Thus, the compressed gas may be recycled notably for the implementation of the displacement step(s)). Similarly the solvent or solvents may be recycled (notably to reconstitute an eluent).

(67) The stationary phase of the invention comprises at least one material chosen from activated alumina, silica gel, activated charcoal, silicic acid, magnesium oxide, calcium hydroxide magnesium hydroxide, cellulose, amylose, a polymerized adsorbent, a derivative thereof, and/or combinations thereof.

(68) The chromatographic separation of the invention may be in the normal phase, i.e. based on polar interactions between the species to be separated and the stationary phase. The stationary phase is then polar in nature, for example a silica phase, functionalized or not.

(69) Alternatively, the chromatographic separation of the invention may be in reverse phase, i.e. based on hydrophobic interactions between the species to be separated and the stationary phase. The stationary phase is then non-polar in nature, for example a silica phase on which hydrocarbon chains are grafted.

(70) Alternatively, the chromatographic separation of the invention may employ a stationary phase of intermediate polarity.

(71) Alternatively, the chromatographic separation of the invention may be a chiral separation. In this case, the stationary phase comprises chiral molecules grafted on a support (for example silica).

(72) The invention may, in particular, be implemented for the separation of non-polar products, preferably on a normal stationary phase. In this case, the product of interest and the impurities engage with the stationary phase non-covalent hydrophilic interactions, notably hydrogen type bonds.

(73) The eluting power of each eluent is, therefore, even stronger when the solvent(s) which constitute it, is or are polar. The product of interest, hydrophobic, is weakly retained on the stationary phase.

(74) Thus, an alkane, or mixture of alkanes, or a mixture of alkane(s) and compressed gas, may be used for the elution step (and for equilibration).

(75) For regeneration, a polar solvent such as ethyl acetate is preferably used to desorb the impurities strongly bound to the stationary phase.

(76) FIG. 3 schematically shows such a method, wherein the graph represents the evolution of the rate of compressed gas in the output stream of a chromatographic column (ordinate) as a function of time.

(77) Phase 1 corresponds to the elution step. The output stream of the column is the eluting eluent, for example an alkane. Phases 2 and 3 correspond to the displacement of the eluting eluent by the compressed gas. The rate of compressed gas in the output stream increases in phase 2 until it reaches a maximum, then remains in a plateau in phase 3. Phases 4 and 5 correspond to the regeneration step, in which a regeneration eluent, for example ethyl acetate, is injected. The rate of compressed gas in the output stream decreases in phase 4 to zero, then remains at zero in phase 5. Phases 6 and 7 correspond to the displacement of the regeneration eluent by the compressed gas. The compressed gas content in the output stream increases in phase 6 until it reaches a maximum, then remains in a plateau in phase 7. Phases 8 and 9 correspond to the equilibration step, in which an equilibration eluent, again for example an alkane, is injected. The compressed gas content in the output stream decreases in phase 8 to zero, then remains at zero in phase 9.

(78) The products of interest to be separated or purified may be, in particular, natural extracts, carotenes, squalenes, vitamins, peptides, insulin (preferably recombinant insulin) or an insulin derivative.