Abstract:
A method of processing a sample comprising introducing a sample into a flow-through device containing a porous solid media therein, and thereafter subjecting the device to microwave energy.

Description:
[0001]    This is a continuation-in-part of U.S. application Ser. No. 10/136,131, filed May 1, 2002. 
     
    
     
       TECHNICAL FIELD  
         [0002]    The invention relates to processing of chemicals in flow-through devices with porous media.  
           [0003]    U.S. Pat. No. 6,139,733, which is hereby incorporated by reference, describes a sample module made of a flow-through device that contains porous media and describes adding a chemical sample to the module prior to connecting the module to (or inserting the module into) a chromatography column. The sample can be added to the module in a dissolution solvent that can be removed by vacuum or heat prior to connection to the chromatography column, and more particularly to.  
         SUMMARY  
         [0004]    In one aspect, the invention features, in general, processing a chemical sample by introducing a sample into a flow-through device containing a porous solid media therein, and thereafter subjecting the device to a radiated energy source such as microwave energy, ultraviolet energy, sonic energy or other means to introduce energy into the device.  
           [0005]    In another aspect, the invention features, in general, introducing a chemical sample into a flow-through device containing a porous solid media therein and active components attached to the solid media, and thereafter subjecting the device to energy in order to accelerate or promote reactions implemented by the active components, the reactions resulting in a reaction product created from the sample.  
           [0006]    In another aspect, the invention features, in general, introducing reagents into a flow-through device containing a porous solid media therein and active components attached to the solid media, causing a chemical reaction involving the reagents in the flow-through device and resulting in a reaction product, thereafter placing the flow-through device into an entrance region within a chromatography column, and thereafter carrying out chromatography on the reaction product.  
           [0007]    In another aspect, the invention features, in general a chromatography sample module including a flow-through member having walls and having an inlet end and an outlet end; a solid porous media disposed within the flow-through member and including attached active components, the media being spaced from the inlet end so that the walls extend above the media and so that the flow-through member defines a receiving region adapted to receive a head piece; and a sample carried on the media.  
           [0008]    In another aspect, the invention features, in general, a tubular member that is sized to fit entirely within the end of a chromatography column containing a separation media, the module having an inlet and an outlet, and solid porous media within the tubular member and spaced from the inlet, so that the tubular member defines a receiving region adapted to receive a head piece. The tubular member is sized to be sealed within the chromatography column with a sealing device used to seal the chromatography column. The solid porous media includes attached active components and carries a sample.  
           [0009]    In another aspect, the invention features, in general a flow-through device having walls and having an inlet end and an outlet end; a solid porous media disposed within the flow-through device including attached active components, the media being spaced from the inlet end so that the walls extend above the media and so that the flow-through member defines a receiving region adapted to receive a head piece; and a sample carried on the media.  
           [0010]    In another aspect, the invention features, in general, a sample module including a tubular member that is sized to fit entirely within the end of a chromatography column containing a separation media, the module having an inlet and an outlet, and solid porous media within the tubular member. The solid porous media includes attached active components and carries a sample.  
           [0011]    In another aspect, the invention features, in general a sample module including a flow-through device having walls and having an inlet end and an outlet end; a solid porous media disposed within the flow-through device including attached active components; and a sample carried on the media.  
           [0012]    Particular embodiments of the invention may include one or more of the following features. In particular embodiments, the sample is introduced into the flow-through device in a solvent that is evaporated by microwave energy prior to carrying out chromatography. In some embodiments, the solid media includes active components attached thereto, and the microwave energy speeds up the reactions involving the active components. In some embodiments the sample includes reagents that undergo a chemical reaction to form a reaction product. The active components attached to the solid media can include scavengers to remove excess reagents. The scavengers can be electrophile scavengers, e.g., amino scavengers, TsNHNH 2  scavengers, or SH scavengers. The scavengers can be nucleophile scavengers, e.g., TsCl scavengers and NCO scavengers. The scavengers can be base scavengers, e.g., a quaternary amine scavenger. The scavengers can be acid scavengers, e.g., TsOH scavengers or COOH scavengers. The active components can be coupling agents, e.g., DCC coupling agents, HOBt coupling agents, or NHS coupling agents. The active components can be a catalyst, e.g., TsOH. The active components can be a catalyst remover, e.g., DEAM.  
           [0013]    Embodiments of the invention may include one or more of the following advantages. The use of microwave energy to evaporate solvent in a flow-through device in which a sample carried in a solvent has been absorbed onto solid media in the flow through device greatly speeds up and simplifies the evaporation process. Attaching active components to the solid media in a flow-through device that can be used to introduce a sample into a chromatography column, permits the same device to be used as a reaction chamber and sample introduction device, simplifying and speeding up synthesis and purification. Subjecting the device with solid media and attached active components to microwave or other energy speeds up the synthesis or other reactions therein.  
           [0014]    Other features and advantages of the invention will be apparent from the following detailed description and from the claims. 
       
    
    
     DESCRIPTION OF DRAWINGS  
       [0015]    [0015]FIG. 1 is a FIG. 1 is a diagrammatic vertical sectional view of a flow-through device with a porous media therein.  
         [0016]    [0016]FIG. 2 is a diagrammatic view showing processing a sample in the FIG. 1 device in a microwave chamber.  
         [0017]    [0017]FIG. 3 is a schematic diagram showing subsequent use of the FIG. 1 device in a chromatography system.  
         [0018]    [0018]FIG. 4 shows how the FIG. 1 device fits within a chromatography column of the FIG. 3 chromatography system.  
         [0019]    [0019]FIG. 5 illustrates reactions that can take place in the FIG. 1 device when it is used as a reaction device.  
         [0020]    [0020]FIG. 6 is a schematic diagram showing subsequent use of the FIG. 1 device in an alternative arrangement in a chromatography system.  
         [0021]    [0021]FIG. 7 is a diagrammatic vertical sectional view of an alternative embodiment of a flow-through device with a porous media therein.  
         [0022]    [0022]FIG. 8 is a diagrammatic vertical sectional view of a second alternative embodiment of a flow-through device with a porous media therein.  
         [0023]    [0023]FIG. 9 is a diagrammatic vertical sectional view of a third alternative embodiment of a flow-through device with a porous media therein. 
     
    
     DETAILED DESCRIPTION  
       [0024]    Referring to FIG. 1, there is shown flow-through device  10 , which includes cylindrical tube  12 , porous plates  14 ,  16  (made of inert plastic porous frits or glass or Teflon), and porous solid media  18  (only partially shown in the figures) between porous plates  14 ,  16 . Tube  12  can be made from glass, polyethylene, polypropylene, Teflon and other plastics. Media  18  can take various forms depending on the application. Media  18  can be silica, other conventional chromatography media, or solid media that has attached active components such as scavengers, coupling agents, catalysts, or catalyst removers.  
         [0025]    Referring to FIG. 2, flow-through device  10 , containing a sample to be processed therein, is shown being subjected to microwave energy in microwave chamber  20 . In some applications, the processing involves removal of a dissolution solvent in which a sample compound of interest is dissolved. In other applications, the media plays an active role in chemical reactions taking place in the flow-through device. In some applications a conventional microwave oven can be used as the microwave chamber. In some other applications, it is better to use a microwave chamber with more precise controls, e.g., units available from Personal Chemistry, CEM or Milestone, Inc. (Monroe, Conn.).  
         [0026]    Use of Flow-Through Device  10  for Removal of Dissolution Solvent  
         [0027]    As is described in the above-referenced patent, when chemists optimize liquid chromatographic separations conditions, they may need to dissolve the sample mixture in a dissolution solvent which may be nonideal for elution. This can result in poor separation and poor recovery of desired components in a chromatography column. For example, polar solvents such as methanol, isopropanol (IPA), acetone, and ethylacetate (EtOAc) can interfere with chromatographic purification. The above-referenced patent describes adding a sample dissolved in a dissolution solvent to the top of the flow-through device (referred to as a sample module in the patent), where it is drawn into the media by capillary action. The sample absorbs onto the media, and the dissolution solvent is then removed by placing the flow-through device in a vacuum chamber and/or applying heat prior to placing the device in, or otherwise connecting it to, a chromatography column.  
         [0028]    In order to avoid the use of a vacuum chamber or heat and to accelerate the drying of the solvent, one can instead subject the sample to microwave energy in microwave chamber  20 . For example, subjecting flow through devices available from Biotage under the Flash 12 trade designation and containing one ml of the solvents IPA, EtOAc, acetone, methanol, and dichloromethane (DCM) in a conventional microwave oven (power set at 30) for 60 seconds resulted in the following percentage evaporations respectively, 82%, 72%, 96%, 88% and 92%. In general, removal of 80% of the polar solvent eliminates the interference of the chromatographic separation. When one is using the microwave chamber and sample module solely for the purpose of removing a dissolution solvent prior to chromatography, one may wish to use an inert media (e.g., sea sand or diatomaceous earth) instead of silica, in order to minimize the possibility of hydrolyzing acid sensitive groups. When polar solvent is removed, sample retention is enhanced, compound resolution is improved and tighter elution bands result. There also are increased separation efficiencies, lower volume fractions and increased loading capacities.  
         [0029]    Referring to FIG. 3, flow-through device  10  (with a preabsorbed sample therein) is used in chromatography system  30 , which also includes a source of solvent  32  (different than the polar dissolution solvent), pump  34 , liquid chromatography column  38 , and sample fraction collection system  40 . In this system, solvent from source  32  is pumped by pump  34  through flow-through device  10  and chromatography column  38 , carrying sample from device  10  thereto, to perform the chromatographic separation of the sample. FIG. 4 shows how flow-through device  10  is sized to fit entirely within the end  42  of chromatography column  38  containing a separation media  44 . In device  10 , the upper plate  14  is spaced from the upper end so that tubular member  12  defines a receiving region adapted to receive the lower end  45  and the lower compressible sealing ring  46  of sealing head piece  48 , which also has an upper compressible sealing ring  50  for providing a seal to the chromatography column  38 .  
         [0030]    Alternatively, instead of inserting the device  10  into chromatography column  38 , device can be placed in a remote holder  70  and connected to the chromatography column by a solvent tube  72 , as shown in FIG. 6. Solvent could also be added to device  10 , which is then placed directly into column  38 , or remote holder  70  connected to chromatography column  38  by tube  72 .  
         [0031]    Device  10  can also be implemented in different forms, as shown in FIGS.  7 - 9 .  
         [0032]    [0032] 90  on the top edge which eases insertion of both lower and upper frits  84 ,  88 . In this case where the upper porous frit  88  is below the top of the tube  82 , frit  88  may or may not form Referring to FIG. 7, flow-through device  80  includes short plastic tube  82 , which has a lower inert plastic porous frit  84  inserted so as to be flush with the bottom of tube  82 . Tube  82  is then filled with a solid support  86  (e.g., porous media) to a pre-determined fill level, and a second inert plastic porous frit  88  is inserted. In this embodiment this top frit  88  is not flush with the top of the tube  82  and holds the solid support  86  in a stable form during shipping and ensures “plug flow” during use. In this embodiment the tube has a chamfer a sealing region  92  to allow a sealing head to be inserted which may or may-not be in contact with the top frit.  
         [0033]    Referring to FIG. 8, flow-through device  100  includes short plastic tube  102 , which has a lower inert plastic porous frit  104  inserted so as to be flush with the bottom of the tube  102 . Tube  102  is then filled with a solid support  106  (e.g., porous media) to a pre-determined fill level, and a second inert plastic porous frit  108  is inserted. In this embodiment this top frit  108  is flush with the top of the tube  102  and holds the solid support  106  in a stable form during shipping and ensures “plug flow” during use.  
         [0034]    Referring to FIG. 9, flow-through device  110  includes a longer plastic tube  112 , which has a lower inert porous frit  114  inserted so as to be flush with the bottom of the tube  112 . The tube is then filled with a solid support  116  (e.g., porous media) to as pre-determined fill level, and a second inert plastic porous frit  118  is inserted. In this embodiment this top frit  118  is not flush with the top of the tube  112  and holds the solid support  116  in a stable form during shipping and ensures “plug flow” during use. In this embodiment the tube  112  has a liquid receiving region  120  to enable wash solvent to be added after the liquid reaction has been absorbed.  
         [0035]    Use of Flow-Through Device  10  as a Reaction Chamber  
         [0036]    Flow-through device  10  can also be used as a reaction chamber in which the solid media includes attached active components such as scavengers, coupling agents, catalysts, or catalyst removers that assist in a chemical reaction therein. In this application, device  10  serves as a reaction chamber for solid phase organic synthesis (SPOS) or solid-assisted synthesis (SAS). In typical SPOS, a desired product (e.g., a small organic molecule being created as part of a combinatorial library) is synthesized on a bed; reactants and excess reagent stay in solution, and, at the end of the synthesis process, the excess reagents are washed out. In typical SAS, solid supports are used to hold reagents, catalysts for synthesis or chemoselective scavengers used to remove excess reactants during purification; this approach when applied to solution phase typically requires a long time for completion and involves many manual steps including washing and extractions.  
         [0037]    An example in which device  10  is used to facilitate scavenging of excess reagents is shown in FIG. 5. In this example, reagent A and reagent B are introduced into a flow-through device  10  that includes solid media  18  with attached nucleophile scavengers N and attached electrophile scavengers E. Reagents A and B combine to form the Product, and excess reagent A and excess reagent B are removed by the scavengers, resulting in a Purified Product, which is removed from device  10  in liquid form. The reaction can take place at room temperature or be aided by application of microwave energy (in microwave chamber  20  in FIG. 2) or conventional heat (e.g., from a hot plate) or a UV lamp. The use of microwave energy is superior because it results in an extremely short reaction time.  
         [0038]    In a reaction arrangement where, following synthesis, the desired product is purified in a chromatography column, flow-through device  10  provides for ease of introduction of the sample into the chromatography column as described in the above-referenced patent. Where it is desired to remove solvent prior to purification in the chromatography column, microwave energy can also be used to provide fast solvent evaporation. By using microwave synthesis on chemical samples and/or reagents in flow-through device  10  (with or without microwave drying) and then directly connecting device  10  to chromatography column  38  for separation and purification, one can potentially synthesize and purify new compounds in less than one hour.  
         [0039]    Examples of nucleophile scavengers N are TsCl scavengers and NCO scavengers. These scavengers can be used to remove excess nucleophiles including amine, hydrazine, alcohols and organometallics.  
         [0040]    Examples of electrophile scavengers are amino scavengers, TsNHNH 2  scavengers, and SH scavengers. The amino scavengers can scavenge acid chloride, sulfonylchloride and isocyanates. The TsNHNH 2  scavengers can scavenge aldehydes and ketones. The SH scavengers can scavenge alkylating agents, ranging from octyl bromide to benzyl bromide. Other electrophile and nucleophile scavengers can be used.  
         [0041]    In addition, base scavengers, e.g., quaternary amine, can be used as a general base to quench reactions, neutralize amine hydrochlorides or to scavenge a variety of acidic molecules like carboxylic acids or acidic phenols.  
         [0042]    Also, acid scavengers, e.g., TsOH and COOH, can be used. E.g., solid media with attached TsOH can be used as an equivalent to the strong cation-exchange resin, Amberlyst A-15 (Rohm &amp; Hass). The device  10  with TsOH attached to the solid media can be used for removal of basic compounds, e.g., primary, secondary and tertiary amine, by quaternary salt formation. Also it can be used for quenching reactions with aqueous or soluble organic acids and for Boc-deblocking by catch and release of amine derivatives.  
         [0043]    Coupling agents, such as DCC, HOBt and NHS, can also be attached to solid media and used for the synthesis of amides and esters.  
         [0044]    A catalyst, e.g., TsOH can also be attached to a solid media and used as a catalyst for esterification.  
         [0045]    A catalyst remover can also be attached. E.g., DEAM attached to a solid media is highly efficient in scavenging oxopilic inorganic and organometallic complexes, including those of boron, titanium and tin. This resin can be used to quench reactions and remove metallic reagents, catalysts or byproducts.  
         [0046]    In addition to synthesis reactions, sample module  10  can be used to carry out other reactions, e.g., one or more of the following reactions:  
         [0047]    i. Organometallic nucleophilic additions (e.g. Grignards, organocuprates, lithiates, etc.)  
         [0048]    ii. Electrophilic additions to carbon-carbon multiple bonds.  
         [0049]    iii. Sigmatropic rearrangements.  
         [0050]    iv. Cycloadditions.  
         [0051]    v. Thermal eliminations.  
         [0052]    vi. Reductions (including hydrogenations).  
         [0053]    vii. Oxidations.  
         [0054]    viii. Multi-component condensations.  
         [0055]    ix. Functional group interconversions.  
         [0056]    x. Unimolecular rearrangements.  
         [0057]    xi. Reactions involving transition metals.  
         [0058]    xii. Aromatic substitutions.  
         [0059]    xiii. Free-radical reactions.  
         [0060]    xiv. Reactions of carbonyl compounds.  
         [0061]    xv. Nucleophilic substitution reactions.  
         [0062]    The reactions already described, including those involving the various scavengers, coupling agents, catalysts and catalyst removers, can be promoted and accelerated by placing the device  10  with the indicated solid media and reagents in microwave chamber  20  and applying microwave energy. In addition, the efficiencies of the reactions are improved such that the amount of excess reagents needed can be reduced. Alternatively, the device can be subjected to other forms of energy, including other forms of radiated energy, to promote and accelerate the reactions.  
         [0063]    Use of flow-through device  10  as described can eliminate the manual manipulation involved in cleaning up a sample through extractions and washing and also provides a convenient reaction vessel.  
         [0064]    Other embodiments of the invention are within the scope of the appended claims.