Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
  • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
  • B01D15/1814—Recycling of the fraction to be distributed
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
  • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
  • B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/26—Conditioning of the fluid carrier; Flow patterns
  • G01N30/38—Flow patterns
  • G01N30/44—Flow patterns using recycling of the fraction to be distributed
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
  • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
  • B01D15/1814—Recycling of the fraction to be distributed
  • B01D15/1857—Reactive simulated moving beds

Definitions

• the invention relates to processes of performing preparative chromatography in an efficient, repetitive manner, as well as to apparatus therefor.
• Feed is continuously injected into the interior of the SMB profile; extract and raffinate are continuously collected; and fresh mobile phase is also added continuously.
• the entire profile travels around the system. For example, in one possible configuration of a 16 column array, the feed is injected between columns 7 and 8; the mobile phase is injected between columns 16 and 1 ; the raffinate is collected between columns 1 1 and 12; and the extract is collected between columns 3 and 4.
• the profile moves to the right, all the injection and collection points are switched simultaneously one column to the right.
• the feed point is between columns 9 and 10; the mobile phase point, between columns 1 and 2; the raffinate point between columns 12 and 13; and the extract point, between columns 4 and 5.
• the switching occurs periodically at the appropriate times. Because the fluids injected through the feed and mobile phases points are collected at the raffinate and extract points, a steady state develops.
• a further characteristic of SMB chromatography is that there are four flow rates across the profile. It is also important to stress that classical SMB chromatography is truly continuous: the mobile phase and feed pumps never stop pumping material into the system; and the extract and raffinate lines never stop delivering collected purified material.
• the prior art also contains three patents that are relevant. USP 4,267,054, USP 4,379,751 , and USP 4,970,002 However, the processes of these patents use closed-loop recycling.
• An object of the invention is to provide economical and efficient HPLC processes. Another object is to provide apparatuses for such processes.
• one aspect of the invention is to employ recycling techniques coupled with the periodic injection of fresh samples.
• Another aspect of the current invention resides in the use of a second solvent pump (referred to as Pump 2 in the attached figures) that prevents the chromatographic profile from stalling during the collection of certain fractions. This feature greatly increases the productivity of the process.
• Pump 2 a second solvent pump
• the employment of two pumps for two columns is itself a novel and useful subcombination.
• a detector is employed, although a detector is not essential for the performance of the invention.
• the entire process becomes visual and intuitive, greatly facilitating methods development.
• the detector allows the use of software in the initiation of all collection and control events such as the collection of fractions, the injection of fresh sample, column switching, pump control, etc.
• software determines the correct point on the chromatographic profile to initiate a given event.
• the software also permits collection and control events to be timed events. For example, the detection of the ascending slope coupled with a timer leads to a very satisfactory process.
• the mobile phase pump can be switched off during injection. This prevents the mixing of the fresh sample with the partially separated profile and results in a significantly improved separation. This technique is particularly useful in difficult separations.
• the mobile phase pump can be left on during injection.
• a mechanism is provided to generate a pulse of strong solvent to elute later eluting fractions more quickly and to concentrate these fractions.
• features of the apparatus comprise: two preparative chromatographic columns, two 4-way valves used for column switching, at least one and preferably two solvent pumps (Pumps 1 and 2), an injection pump (or system), and a preferably 3-way recycle valve used to send the mobile phase to waste or to recycle it to Pump 1.
• solvent pumps Pumps 1 and 2
• injection pump or system
• a preferably 3-way recycle valve used to send the mobile phase to waste or to recycle it to Pump 1.
• Any type of preparative chromatographic column can be used.
• Each column is preferably packed with the same type and amount of stationary phase or an equivalent thereof. For best results, the columns should have nearly the same efficiency as measured by the number of theoretical plates
• control events including, but not limited to, valve switching, toggling on and off of pumps, and adjusting pump flow rates, are initiated by the software solely on the basis of time
• the progress and success of the separation is determined by periodic and/or on-line sampling of fractions followed by analysis of the fractions by analytical instruments not associated with the preparative chromatographic process
• the process can be used to perform any type of chromatographic separation including, but not limited to, normal phase chromatographic separations, reverse phase chromatographic separations, chiral chromatographic separations, ion exchange chromatographic separations, affinity chromatographic separations, and size exclusion chromatographic separations
• Figure 1 is a generalized schematic flowsheet of the invention
• Figure 2 is another flowsheet of the invention differing from Figure 1 by the use of a 6-way sequential valve
• Figure 3 is a chromatogram of Example 1
• Figure 4 is a graph of cycle 6 of Example 1 ,
• FIG. 5 is a chromatogram of Example 2
• FIG. 6 is a chromatogram of Example 3,
• Figure 7 is a graph of cycle 3 of Example 3
• Figure 8 is a chromatograph of Example 4
• Figure 9 is a schematic flowsheet of the system used in Example 5, having a third 4-way valve instead of the 6-way sequential valve in Figure 2, and also provided with a more sophisticated fraction collection system,
• Figure 10 is a chromatogram of Example 5
• Figure 1 is a graph of cycle 4 of Example 5
• Figure 12 is a schematic flowsheet of the invention, similar to Figure 1 , but depicting a 3-way valve and a generalized fraction collection system,
• Figure 13 is similar to Figure 1 , but wherein the fraction collection system is a 2-way collection valve system
• Figure 14 is a schematic flowsheet, similar to Figure 1 , wherein the fraction collection system is a series of high pressure 3-way valves,
• Figure 15 is a schematic flowsheet of a modification of Figure 1 , wherein pump 2 is employed for injection of the sample, thereby eliminating the injection pump
• Figure 16 is a schematic flowsheet similar to Figure 1 , but employing a calibrated injection loop
• Figure 17 is similar to Figure 2, but employing the fraction collection system of Figure 9
• the abbreviation "PT" in Figure 1 and in other figures stands for "pressured transducer”
• the abbreviations "P1 P5" stand for a valve setting permitting air to be introduced and the abbreviations "V1 V5" stand for a valve setting for collecting the fraction
• Figure 1 shows a generalized schematic diagram of the invention Virtually any type of fraction collection system used in preparative chromatography can be used to collect purified product. Methods of automated sample injection other than the simple injection pump shown are also possible
• the two 4-way valves serve to switch the profile onto the appropriate column They will switch simultaneously This event will be a timed event
• Pumps 1 and 2 are mobile phase pumps
• the flow rate of Pump 1 is entered into the program at the beginning of the run Pump 1 can be switched off during injection This is useful because it prevents mixing of the fresh sample with the partially separated profile, thus resulting in a better separation
• This switching off of Pump 1 during injection could be a timed event, or it could be triggered by the relevant parameters determined by the system and software
• Pump 1 can pump either solvent A or solvent B
• Solvent A is the separating solvent and is the mobile phase that has been determined to give the best separation
• Solvent B is a stronger solvent, e g , 100% methanol as compared to an 80 20 by volume methanol water solution
• a short pulse of solvent B can be used to wash the more retained components off the relevant column to be collected by the fraction collection system or rejected to waste (By "pulse” is meant a momentary injection of solvent B, lasting about 1-10% of the time of complete cycle for example ) This pulse of stronger solvent reduces the time needed to collect these fractions and also significantly increases the
• Pump 2 The purpose of Pump 2 is to prevent the stalling of the profile. Pump 2 can be turned on during these collection events, causing the profile to continue through the column. Throughput (amount of product collected per unit time) is significantly increased by using Pump 2 to prevent stalling of the profile.
• the 3-way recycle valve is typically set to recycle mobile phase to the inlet side of Pump 1 during the operation of Pump 2. This significantly minimizes the amount of mobile phase used, since otherwise the mobile phase from Pump 2 would go to waste.
• Pump 2 is run at a slightly lower flow rate than Pump 1 . Pump 1 makes up the extra amount of solvent A it needs by drawing it directly from the reservoir.
• the 3-way recycle valve can also be set to recycle pure mobile phase to the inlet side of Pump 1 when Pump 2 is shut off and no fractions are being collected. The columns are then connected in series, and Pump 1 provides the entire impetus to move the profile through the columns. Mobile phase leaving the second column in series can be diverted to waste or recycled to the inlet of Pump 1.
• the 3-way recycle valve is set to waste during injection. This assures that the system will not be "dead headed" during injection.
• the current invention is a repetitive process that can run for hours or days on end. It is, therefore, necessary that it be automated via computer control.
• TurboPrep® control software proprietary software of EM
• Figure 2 shows one of the preferred embodiments of the current invention in which the collection system is a 6-way sequential valve. This embodiment was used to obtain all the results presented in Examples 1-4. Other preferred embodiments using different fraction collection systems and different injection systems are discussed hereinafter.
• the 4-way valves are switched, directing the profile onto the appropriate column. (The 6-way valve is already in position 1 )
• the recycle valve can be set to recycle mobile phase to the inlet of Pump 1 or to send the stream to waste.
• the 6-way valve is switched to position 2 to divert the stream to waste in anticipation of the approach of the profile 3
• the 6-way valve is switched to position 3 to collect fraction 1.
• the injection pump is turned on to pump fresh sample into the interior of the profile During injection the 3-way recycle valve is switched to waste. This assures that the system will not be “dead headed” during injection Also during injection, Pump 1 may be turned off if desired
• the injection pump is turned off If Pump 1 had been turned off during injection, it will restart when the injection pump is turned off
• the recycle valve can be set to recycle mobile phase to the inlet of Pump 1 or to send the stream to waste 7.
• the 6-way valve is switched to position 5 to collect fraction 2.
• Pump 2 can also be turned on to prevent stalling of the profile.
• the 6-way valve is switched to position 6. This stops the collection of fraction 2 and diverts the stream to waste to prevent the circulation of uncollected product and contaminants. During this time, Pump 2 can also be turned on to prevent stalling of the profile.
• the recycle valve can be set to recycle mobile phase to the inlet of Pump 1 or to send the stream to waste.
• the manual inject valve is not an essential feature of the current invention. Its purpose is to allow testing of the columns by injecting small amounts of standard solutions. Figures 3 through 11 will be discussed in detail in connection with the following Examples.
• Figure 12 shows a generalized schematic of a fraction collection system that is connected to the rest of the system via a high pressure 3-way vaive Any type of fraction collection system used in preparative chromatography can be connected in this way
• a sequential fraction collector in which the tubing conducting the fraction is sequentially interfaced with each fraction collection vessel
• an array of valves connected to a central low dead-volume cavity into which the fraction is conducted, any of the valves can be chosen in any order to conduct the fraction further to the collection vessel of choice
• Figure 13 shows another method of collecting fractions in which a linear array of high pressure 2-way valves is connected to the system via a series of closely spaced cross-shaped fittings
• Figure 14 shows another method of collecting fractions in which a series of high pressure 3-way valves are connected to the system in series
• a much larger number of fractions can be collected with any of the examples discussed above and depicted in Figures 12, 13 and 14 than when a 6-way sequential valve (as depicted in Figure 2) is used
• the basic order of operation is the same no matter what type of fraction collection system is used the 4-way valves are switched to select the column order, fractions are collected on the front part of the profile, fresh sample is injected into the interior of the profile, fractions are collected on the back side of the profile
• These illustrations of fraction collection system are not intended to be exhaustive as it is possible to connect other types of fraction collection systems to the current invention Any type of fraction collection system used in preparative chromatography can be used
• Figure 15 shows a method of injecting sample using Pump 2 Using the 3-way valve, either solvent A or the sample solution can be pumped by Pump 2 Another 3-way valve is needed to purge the outlet line of Pump 2 of either solvent A or sample Since sample can be lost during a purge, the preferred method is to use a separate injection pump as depicted in Figures 1 and 2
• Figure 16 shows a method of injecting sample by using a 6-way valve and calibrated loop This method of injection is not as flexible as that depicted in Figures 1 and 2, especially during methods development
• Pump 1 and Pump 2 were ST 140 preparative HPLC pumps from EM Separations Technology
• the flow rate of Pump 1 was set with the software, and the flow rate of Pump 2 was set manually with the thumb wheel on the front of the pump
• the injection pump was an Eldex model B-100-S-4 metering pump
• the flow rate for this pump was set manually with a micrometer Control of Pump 1 , Pump 2, and the Eldex pump was accomplished via software through an Opto 22 interface
• the two 4-way valves, the 6-way sequential vaive and their air-powered actuators were obtained from Rheodyne
• the two 3-way valves were obtained from Mace All valves were air actuated and were controlled via software through the Opto 22 interface
• the detector was obtained from Knauer. It was a variable wavelength U V HPLC detector equipped with a high pressure flow cell.
• Proprietary TurboPrep® software from EM Separations Technology was used to control all pumps and valves.
• the software ran on a 486 computer from Dell Computer Corporation.
• the computer was connected to the system by way of an interface from Opto 22.
• the two columns were annular expansion columns from EM Separations Technology. The dimensions of the columns were 50 mm diameter x 200 mm in length. Each column was packed with approximately 244 g of RP Select B, particle size 12 ⁇ m.
• RP Select B is a C8 octane phase bonded to silica and is a product of Merck KGaA, Darmstadt, Germany.
• the mobile phase was methanol:water 80:20. Both the methanol and the water were HPLC grade and were obtained from EM Science.
• the nominal flow rate for Pump 1 was 70 mL/min. When used, the nominal flow rate for Pump 2 was 65 mL/min.
• the mixture undergoing separation was a solution of methyl and propyl p-hydroxybenzoates.
• the methyl and propyl p-hydroxybenzoates were obtained from Aldrich.
• the sample solution was made by dissolving 30 mg/mL each of methyl and propyl p-hydroxybenzoate in methanol:water 80:20.
• the methyl p-hydroxybenzoate is less retained and eluted first; the propyl p-hydroxybenzoate eluted second.
• the flow rate of the Eldex injection pump was set at 20 mL/min. Each injection occurred for 1.0 minute giving an injection volume of 20 mL.
• Figure 3 shows a chromatogram that illustrates all of the stages of the process The end of the first 20 mL injection occurred at 0 minutes The second injection began at about 5 8 minutes, subsequent injections occurred thereafter at 6 95 minute intervals The flat part in the middle of the profiles is due to the shut-off of Pump 1 during injection flow ceased through the detector resulting in a constant signal from the detector
• Figure 5 shows a chromatogram which illustrates this example As with Example 1 , the end of the first 20 L injection occurred at 0 minutes, and the second injection began at about 5 8 minutes Beginning with cycle 2, specific times were not imposed on any of the events except for the switching of the 4-way valves and the rotation of the 6-way valve to position 2 Both of these events occurred every 10 1 minutes beginning with the second cycle
• Figure 6 shows a partial chromatogram of this example
• Figure 7 shows cycle 3 in more detail. Note that because Pump 1 is never turned off, there is no flat region in the middle of the profiles as in Examples 1 and 2. However, it is also obvious that, beginning with cycle 2, the resolution is worse than in Examples 1 and 2. The resolution could be improved by using any of the techniques discussed above.
• Pump 2 does not turn on during the collection of fraction 2 and during its subsequent waste step.
• the throughput could be significantly improved by using Pump 2, as is shown in the next Example.
• Example 5 Figure 9 shows a schematic diagram of the system used in this example
• the 6-way sequential valve in Figure 2 has been replaced by 4-way valve #3 that leads to an array of fraction collection valves
• any collection valve can be chosen in any order It is therefore, more flexible than the design shown in Figure 2 in which one is constrained to choose collection lines in a specific order
• 4-way valve #3 by ⁇ passes the collection valve array and communicates directly with 4-way valve #1 Note in Figure 9 that 4-way valve #3 connects Pump 2 to the system
• Pump 2 can be turned on to prevent stalling of the chromatographic profile
• 4-way valve #3 has been switched back to the default position
• Pump 2 can be turned on to wash sample from the common line leading to the fraction collection array, thus avoiding contamination of fractions
• Figure 10 shows a chromatogram that illustrates the use of this design in the separation process Note that is very similar to Figure 3, the chromatogram for Example 1 This is to be expected since the separation process is the same we are merely using a different method of collecting the fractions in this Example 5 As with Example 1 , the cycle time for the current example was 6 95 minutes
• FIG 1 1 shows cycle 4, in which steady state has been achieved, in more detail
• the method of collecting fractions in the current example requires more steps than that used in Example 1 To collect a fraction, 4-way valve #3 must be opened, and the collection valves of interest must be opened Also, the common collection line must be washed between fractions to prevent cross contamination However, since the collection valves can be opened in any order and since more collection valves can easily be added, the fraction collection method used in the current example is more flexible than that used in Example 1-4

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• 1995
  • 1995-11-30 US US08/566,425 patent/US5630943A/en not_active Expired - Lifetime
• 1996
  • 1996-11-12 DE DE69631903T patent/DE69631903T2/de not_active Expired - Lifetime
  • 1996-11-12 WO PCT/EP1996/004937 patent/WO1997020206A1/en not_active Ceased
  • 1996-11-12 JP JP52010597A patent/JP4009671B2/ja not_active Expired - Fee Related
  • 1996-11-12 CA CA002238954A patent/CA2238954A1/en not_active Abandoned
  • 1996-11-12 EP EP96939008A patent/EP0877936B1/en not_active Expired - Lifetime

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