WO2016057715A1

WO2016057715A1 - Graphene media for chromatography

Info

Publication number: WO2016057715A1
Application number: PCT/US2015/054553
Authority: WO
Inventors: Siddiqui OZAIR
Original assignee: Nitto Denko Corporation
Priority date: 2014-10-07
Filing date: 2015-10-07
Publication date: 2016-04-14

Abstract

Described herein are methods separating mixtures of organic materials, the methods comprising retaining a graphene oxide; contacting the mixture with a graphene oxide; passing an organic eluent over the graphene oxide to concurrently elute the organic materials in the mixture at different rates; and selectively collecting the at least one organic material from the passed eluent, separating the at least one organic material from another. Also described herein are chromatographic materials, columns and systems incorporated in same methods.

Description

GRAPHENE MEDIA FOR CHROMATOGRAPHY

BACKGROUND

Field

[0001] The present disclosure relates to a stationary phase material for use in chromatography, a column using the same, and a method of separation using the same.

Description of the Related Art

[0002] Chromatography and solid phase extraction relate to any of a variety of techniques used to separate complex mixtures based on differential affinities of components of a sample carried by a mobile phase with which the sample flows, and a stationary phase through which the sample passes.

[0003] When one solute is made relatively immobile (by transitory retention on a stationary phase) and another made relatively mobile (by partitioning into the mobile phase), separation of plural solutes can occur. Chromatography is distinctive from filtering or adsorption, in that each analyte is partitioned according to its retention factor (R_f) and hence eluted at a different interval.

[0004] Chromatography is a method used to separate individual chemical compounds from other compounds in mixtures of compounds. Planar chromatography is a method in which the stationary phase is present as or on a plane. Column chromatography is a method in which the stationary phase is within a tube or cylinder. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling.

[0005] The classical preparative chromatography column is a glass tube with a diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter (a glass frit or glass wool plug - to prevent the loss of the stationary phase) at the bottom. Two methods are generally used to prepare a column: the dry method and the wet method. For the dry method, the column is first filled with dry stationary phase powder, followed by the addition of mobile phase, which is flushed through the column until it is completely wet, and from this point, is never allowed to run dry. For the wet method, a slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles. A solution, usually containing organic material, is loaded or pipetted on top of the stationary phase. This solution is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent. Eluent is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent-filled stoppered separating funnel is attached to the top of the column.

[0006] The individual components are retained by the stationary phase differently and separately while they are running at different speeds through the column with the eluent due to differences in the desired characteristic used to separate the individual components. At the end of the column, the individual components elute one at a time.

[0007] During the entire chromatography process, the eluent can be collected in a series of fractions with the fractions being collected automatically by means of fraction collectors. The productivity of chromatography can be increased by running several columns at a time, in which case, multi-stream collectors can be used. The composition of the eluent flow can be monitored and each fraction analyzed for dissolved compounds, e.g., by analytical chromatography, refractive index, UV absorption, or fluorescence. Colored compounds (or fluorescent compounds with the aid of an UV lamp) can be seen through the column as moving bands.

[0008] The stationary phase or adsorbent in column chromatography can be a solid. The most common stationary phase for column chromatography is silica gel, followed by alumina. However, there are problems with the use of silica gel. Silica gel is expensive, has no industrial scale usability, is slow (particularly with reverse-phase chromatography), and creates a risk of environmental pollution. Thus, there is a need for an alternative stationary phase material. SUMMARY

[0009] As disclosed herein, graphene may be used as a stationary phase for chromatography. Graphene is a one-atom-thick layer of carbon.

[0010] Some embodiments include a method of separating organic compounds, comprising: passing a mobile phase comprising a mixture of organic compounds through a stationary phase comprising a reduced graphene oxide, a graphene oxide, a graphene gel, or a combination thereof.

[0011] In some embodiments, the method of separating at least one organic material from another in a mixture is provided, using a stationary phase comprising a modified graphene; contacting the mixture with the stationary phase to a partially retained organic material with the stationary phase; and passing a mobile phase comprising an organic eluent through the graphene composition to elute the organic materials in the mixture at different rates. In some embodiments, the method may include providing a reservoir, having an inlet and outlet defined therein, creating a dispersion comprising the modified graphene, and disposing the dispersion in the reservoir. In some embodiments, the method may include providing a substrate and disposing the modified graphene on to the substrate to be used as a stationary phase in chromatography. In some embodiments, the method may further comprise selectively collecting organic material from the passed eluent and separating a specific organic material from another.

[0012] In some embodiments, the graphene may comprise a graphene oxide. In some embodiments, the modified graphene may be reduced graphene oxide, graphene oxide, and/or graphene gel. In some embodiments, the unfunctionalized graphene oxide may be:

COOH COOH

[0013] In some embodiments, the organic eluent may comprise a non-polar solvent. In other embodiments, the organic eluent may comprise hexane, ethyl acetate and/or combinations thereof. [0014] The graphene compositions described herein may be utilized in a variety of applications, including, but not limited to, chromatography gels, chromatography columns and chromatography systems.

[0015] Some embodiments include a chromatography media for transitory retention of a material thereon, comprising a gel comprising a reduced graphene oxide, a graphene oxide, or a combination thereof. Some embodiments include a chromatography column comprising this chromatography media.

[0016] These and other embodiments are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a schematic for a method of separating a mixture into component parts through chromatography with graphene oxide.

[0018] FIG. 2 shows a graphical representation of time points used in the calculation of R_f.

[0019] FIG. 3A is a schematic for a method of separating at least two components from a mixture through a graphene stationary phase.

[0020] FIG. 3B is a schematic depicting the relative distance, rate, or retention factors of differing mixture components through a graphene stationary phase.

[0021] FIG. 4A is a schematic for a method of separating components utilizing a graphene stationary phase and a graphene dispersion.

[0022] FIG. 4B is a schematic depicting the relative distance, rate, or retention factors of different mixture components through a graphene stationary phase and a reservoir.

[0023] FIG. 5A is a schematic for a method of affixing a graphene stationary phase to a substrate.

[0024] FIG. 5B is a schematic depicting the relative distance, rate, or retention factors of different mixture components through an affixed graphene substrate.

[0025] FIG. 6 shows a chromatogram for the flash chromatography normal phase separation as described herein in Example 1 .

[0026] FIG. 7 shows a chromatogram for the flash chromatography normal phase separation as described herein in Example 2. [0027] FIG. 8 shows a chromatogram for the flash chromatography normal phase separation as described herein in Example 3.

[0028] FIG. 9 shows a chromatogram for the flash chromatography normal phase separation as described herein in Example 4.

[0029] FIG. 10 shows a chromatogram for the flash chromatography normal phase separation as described herein in Example 5.

[0030] FIG. 1 1 shows a chromatogram for the flash chromatography normal phase separation as described herein in Example 6.

DETAILED DESCRIPTION

[0031] The term "inert" includes a substance or species that does not facilitate or effect one or more chemical reactions involving that substance or other substances coming in contact with the substance or species.

[0032] The term "modified graphene media," as used herein, includes a chromatographic separatory media that includes graphite oxide, graphene, graphene oxide, or closely related carbon materials for the chromatographic separation of organic materials.

[0033] The term "eluate" refers to the analyte contained in a mobile phase leaving the column, propagating over a medium, or along a plane.

[0034] The term "eluent" refers to the solvent that carries the analyte.

[0035] The term "stationary phase" refers to one of the two phases forming a chromatographic system. The stationary phase refers to the phase that is fixed or substantially fixed within the chromatography system.

[0036] The term "mobile phase" refers to a fluid which moves through or along the stationary phase in a definite direction.

[0037] The term "analyte", as used herein, refers to a substance to be recoverably separated from a mixture during chromatography.

[0038] The term "partially retained" refers to a substance that is eluted at different times during chromatography as compared to other analytes.

[0039] The term "chromatography" refers to a physical method of separation that distributes components between at least two phases: a stationary and a mobile phase. [0040] The term "adsorptive separation" refers to a separation based mainly on differences between the adsorption affinities of the sample components for the surface of an active solid. The sample components may be substantially and or wholly retained on the stationary phase based on the sample's adsorption affinities.

[0041] The term "partition separation" refers to separation of sample components based mainly on the differences between the solubility of the components in the stationary phase, or on differences between the solubility of the components in the mobile and stationary phases (liquid chromatography).

[0042] The term "normal phase chromatography" refers to an elution procedure in which the stationary phase is more polar than the mobile phase.

[0043] The term "reversed-phase chromatography" refers to an elution procedure in which the mobile phase is more polar than the stationary phase.

[0044] The term "dispersion" refers to a mixture of different materials, including, but not limited to, emulsions, solution mixtures, and solid liquid suspensions.

[0045] The term "gradient elution" refers to a procedure in which the composition of the mobile phase is changed continuously or stepwise during the elution process so that sample components are eluted at different gradient points based on the sample components characteristics.

[0046] The term "isocratic elution" refers to a procedure in which the composition of the mobile phase remains constant during the elution process.

[0047] The term "stepwise elution" refers to an elution process in which the composition of the mobile phase is changed in steps during a single chromatographic run.

[0048] The term "retention time" refers to the time between introduction of the sample into the chromatography system and detection of the analyte in the eluate.

[0049] The term "chromatogram" refers to a graphical or other presentation of detector response, concentration of analyte in the eluate, or other quantity used as a measure of analyte concentration versus eluate volume or time.

[0050] The term "selectivity factor" refers to the ratio of the retention factor (R_f) of a first compound to the retention factor of a second compound. [0051] Described here in are methods of separating at least one organic material from another in a mixture. In some embodiments, the method may comprise providing a stationary phase with a modified graphene; contacting the mixture with the stationary phase to partially retain at least one organic material with the stationary phase; and passing a mobile phase comprising an eluent through the modified graphene to elute the organic materials in the mixture at different rates.

[0052] The method may be used to separate biological molecules as well as small organic molecules, such as molecules having a molecular weight of about 1000 Da or about 500 Da or less. In some embodiments, the method is used to separate a first organic compound having a molecular weight of less than about 1000 Da or about 500 Da from a second organic compound having a molecular weight of less than about 1000 Da or about 500 Da.

[0053] In some embodiments, the method may provide for a stationary phase with a reservoir, having an inlet and outlet defined therein; creating a dispersion comprising a modified graphene; and disposing the dispersion into the reservoir. In some embodiments, the method may provide for a stationary phase comprising a substrate having a surface and disposing a modified graphene on the surface. In some embodiments, the method may further comprises selectively collecting one organic material from the passed mobile phase and separating one organic material from another. In some embodiments, the modified graphene comprises a graphene oxide. In some embodiments, the functional ized graphene oxide may be reduced graphene oxide, graphene oxide, or graphene gel. In some embodiments, the functionalized graphene oxide may be:

and/or

In some embodiments, the graphene gel may be:

[0054] In some embodiments, the modified graphene may be reduced graphene oxide, unfunctionalized graphene oxide, graphene gel, or combinations thereof. In some embodiments, the modified graphene comprises a graphene oxide analogue consisting essentially of carbon, hydrogen, and oxygen. In some embodiments, the modified graphene may be functionalized to include at least one reactive oxygen functional group. In some embodiments, the modified graphene may have a surface that is configured to come in contact with a fluidized sample mixture, such as a hydrocarbon.

[0055] In some embodiments, the modified graphene oxide may have an oxygen content of at least about 10%; at least about 15%; at least about 20%; at least about 25%; at least about 30%; at least about 35%; at least about 40%; at least about 45% at least about 50%, or any other value bound by these ranges. In an embodiment, the oxygen content may represent the atomic percent of oxygen atoms present in the modified graphene oxide as approximately 70% to about 90% carbon to approximately 30% to about 10% oxygen corresponding to an atomic ratio of carbon to oxygen of approximately about 10:1 to about 2.33:1 .

[0056] In some embodiments, the graphene oxide consists essentially of carbon, oxygen and hydrogen. In some embodiments, the graphene oxide may be any or all of the following:

reduced graphene oxide (rGO),

graphene oxide (GO),

i 'OOi ! COOH

In some embodiments, modified graphene may comprise a graphene gel. In some embodiments, the graphene gel may comprise at least two graphene cores and a linker element disposed between and covalently linking the two graphene cores. In some embodiments, the graphene gel may be represented by the following formula:

G1 -U-G2 (Formula 1 )

wherein G1 and G2 are independently selected from the modified graphenes described herein, and Li is a linker moiety.

[0057] In some embodiments, the graphene oxide may be cross-linked by a linker, L1 . In some embodiments, the linker L1 may be a polymer (e.g., a polyallylamine) to form a cross-linked modified graphene. Such a cross-linked oxide may also be used as a starting material to form a graphene derivative described herein. In some embodiments, the modified graphene elements, G1 and/or G2 may comprise a reduced graphene oxide and/or an unfunctionalized graphene. In some embodiments, the linker element can comprise any organic group. In some embodiments, the linker element may comprise functionalized carbon chain[s], ether linkages, sulfur linkages, or combinations thereof.

[0058] In some embodiments, the graphene gel may comprise:

wherein n = 1 -100, m = 1 -100.

[0059] In some embodiments, the modified graphene may be a particle, such as a particle having an average particle size between about 1 nm to about 500 nm; about 10 nm to about 450 nm; about 25 nm to about 425 nm; about 50 nm to about 400 nm, or any other values bound by these ranges. In some embodiments, the modified graphene may have a surface area (BET) of about 350 m²/g to about 600 m²/g, of about 375 m²/g to about 550 m²/g, about 420 m²/g to about 525 m²/g, and any other value bound by these ranges. A suitable modified graphene can be reduced graphene oxide (rGO) sold by Graphenea (Gipuzkoa, Spain).

[0060] In some embodiments, the stationary phase may include a substrate and a modified graphene affixed to the substrate. For example, the substrate may be a common industrial or household surface on which a dispersion may be directly applied. Substrates can include, but are not limited to, glass (e.g., slides, thin layer chromatography substrates, windows, mirrors, etc.), resin materials, or plastics (e.g., but not limited to, polycarbonates (PC), polypropylenes (PP), polyethylenes [e.g., polyethylene {PE}, polyethylene terephthalates {PET}, polytetrafluoroethylenes {PTFE}, polyvinylidene fluorides, polyimides and polyamide-imides, perfluoralkoxy polymer resins, fluorinated ethylene propylene (FEP), ethylene tetrafluoroethylene (ETFE), polyesters, polyurethanes), and/or other polymeric surfaces.

[0061] In some embodiments, the stationary phase may comprise a binder to affix the modified graphene to the substrate. In some embodiments, the binder may be polymeric. In some embodiments, the binder may be inorganic. In some embodiments, the binder may be, but is not limited to, gypsum, (e.g., CaSO₄-½ H₂O or Plaster of Paris) or silica acid (Merck KGaA, Darmstadt, Germany). In some embodiments, the polymeric binder can be about 1 -2% by weight.

[0062] In some embodiments, the stationary phase may comprise about 10-15% binder and 85-90% modified graphene. For example, to coat five 20 x 20 plates, about 30 g graphene gel and 60 ml_ water would be needed. In some embodiments, a stationary phase may include polyvinyl alcohol or polyvinylpyrrolidone.

[0063] In some embodiments, the active portion of the stationary phase, e.g. the portion of the stationary phase that excludes any substrate or binder, may be at least about 50%, about 80%, about 90%, or about 95%, reduced graphene oxide, a graphene oxide, a graphene gel, or a combination thereof, by weight.

[0064] In some embodiments, the stationary phase the stationary phase contains low levels of metals such as gold or nickel, such as less than 20%, less than 10%, less than 5%, or less than 1 % by weight of a metal, such as gold or nickel.

[0065] In some embodiments, the eluent may comprise a non-polar solvent. In some embodiments, the eluent may comprise hexane. In some embodiments, the eluent may comprise a polar solvent. In some embodiments, the eluent may water, acetonitrile, methanol, dimethylformamide (DMF), or combinations thereof. In some embodiments, a chromatography media for transitory retention of a material may be a gel comprising a modified graphene. In some embodiments, the modified graphene used in the chromatography media for transitory retention maybe reduced graphene oxide, graphene oxide, or combinations thereof. In some embodiments, the method may comprise a chromatography column and a chromatography system comprising a modified graphene gel.

[0066] Embodiments of the method may comprise using a chromatographic media comprising a modified graphene (referred to hereafter as "chromatographic media") that may separate organic materials from one another in a mixture. In some embodiments, the chromatographic media may be an inert chromatographic media. In other embodiments, the inert chromatographic media may comprise a catalytically-inert graphene oxide or graphite oxide. In some embodiments, the chromatographic media may have, but is not limited to, one or more of hydrogen groups (H), hydroxyl groups

(OH), carbonyl groups (C=O), epoxide

), carboxylic acid (-COOH) groups, or combinations thereof. In an embodiment, the media may have, but is not limited to, one or more species (or "surface moieties") selected from a hydroxyl group, epoxide group, ketone group, ether group, carboxylic acid, carboxylate group, or combinations thereof. In other embodiments, surface moieties may be disposed on the surface of the chromatographic media at various interactive sites. In some embodiments, an interactive oxygen functional group may be an hydroxyl, epoxide, ketone, carbonyl, carboxyl group, or combinations thereof.

[0067] The retention of the analyte may be described as the distribution of the analyte between the stationary phase and the mobile phase. The distribution of a solute between the mobile and stationary phases in chromatography is described by κ, the partition coefficient, defined by K=C_s/C_m, where C_s is the concentration of solute in the stationary phase and C_m is the concentration of the solute in the mobile phase.

[0068] In some embodiments, the chromatographic media may separate organic materials with relatively close retention times. The retention factor (R_f) is determined by measuring the retention time of the analyte (t_R), subtracting the transit time of the mobile phase to pass through the column (t_M), and dividing the difference by t_M, as represented by the formula Rf = (t_R - t_M)/tivi_. FIG. 2 shows a graphical representation of the time points, namely t_R and t_m, needed during elution in order to calculate R_f. In some embodiments, the chromatographic media may separate organic materials having retention factors closer than about 0.5, about 0.25, about 0.2, or about 0.1 relative each other. One exemplary method of testing the R_f may be by determining the R_f of different organic compounds at 25 °C using the same eluent, e.g., 90% hexane/10% ethyl acetate and comparing the ascertained R_f.

[0069] In some embodiments, the chromatographic media may separate a first organic material and a second organic material that have a selectivity factor of 1 when eluted with silica gel. In some embodiments, compounds having no difference in R_f when eluted with silica gel may have a difference of R_f when eluted with the chromatographic media of about 0.1 , about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 .0, about 2.0, about 3.0, about 4.0, about 5.0, or any combination of values within the range.

[0070] In some forms of chromatography, the analyte can be recovered from the stationary phase. To recover the analyte and refresh the stationary phase, in some embodiments, the eluent may comprise a releasing agent. For example, a releasing agent may include, but is not limited to, a high-salt solution or an acidic/basic eluent. In some embodiments, the chromatographic media may be refreshed without the use of an additional releasing agent to recover the analyte. In some embodiments, the chromatographic media may be refreshed by passing sufficient eluent over the chromatographic media to flush the analyte away.

[0071] In some embodiments, the chromatographic media may be reusable. In some embodiments, when a sample has been eluted such that the desired organic material has been partially retained by adsorption onto the chromatographic media, the chromatographic media may be restored by removing the adsorbed organic material. In some embodiments, no additional agent may be necessary to desorb the material from the chromatographic media. In some embodiments, the chromatographic media may be restored by passing sufficient eluent through the system so that the organic material that was partially retained passes through the system. In some embodiments, sufficient eluent to restore the chromatographic media may be about 1 column volume (CV), about 1 .5 CV, about 2 CV, about 2.5 CV, about 3 CV, about 3.5 CV, about 4 CV, about 4.5 CV, about 5 CV, about 6 CV, about 7 CV, about 8 CV, about 9 CV, about 10 CV, or any other any combination of values within the range. In some embodiments, sufficient eluent to restore the chromatographic media can be any combination of the aforementioned values, up to about 100 CVs. In some embodiments, a reusable media can be flushed with an eluent, mild acid and/or mild base and provide within 75%, 80%, 90% of the prior mobility/separation ability.

[0072] In an embodiment, the chromatographic media may remain active even when it is highly loaded. In some embodiments, the chromatographic media can remain active while separating at least about 10 mg/ml of analyte, at least about 100 mg/ml of analyte, and/or at least about 500 mg/ml of analyte. In some embodiments, the chromatographic media may separate about 42 μΙ_ of diisopropylphenol (DIPP) from about 40 μΙ_ of 4-(dibutylamino)benzaldehyde (DBABA). In some embodiments, the chromatographic media can separate about 420 μΙ_ of DIPP from about 400 μΙ_ of DIBA. In some embodiments, the chromatographic media can separate 400 mg of X from 400 mg of Y, where X and Y are organic sample components. Without wishing to be bound by any theory, it is believed that a graphene derivative, such as a graphene derivative containing a metal ion or a protein, can have a significantly higher analyte loading capacity than a conventional stationary phase material when used in chromatography. As another example, the graphene derivative may have an analyte-loading capacity of at least about 60 mg/mL, at least about 80 mg/mL, at least about 100 mg/mL, at least about 150 mg/mL, at least about 200 mg/mL, at least about 300 mg/mL, at least about 400 mg/mL, at least about 500 mg/mL, or any other loading capacity bound by these vaules of the graphene derivative measured by an analysis. By contrast, the analyte- loading capacity of a conventional stationary phase material for chromatography can be at most about 60 mg/mL.

[0073] While not being bound by any particular theory, the partial retention of the analyte may be primarily explained by temporary chemical interaction, e.g., non- covalent interactions like ionic interaction, hydrogen bonds, Van der Waals forces between the analyte and the functional groups of the chromatographic media. Additionally, some amount of analyte may be minimally retained by adsorption onto the chromatographic media. In some embodiments, the chromatographic media can adsorb at least about 0.001 wt%, at least about 0.01 wt%, at least about 0.05 wt%, at least about 0.1 wt%, at least about 0.5 wt%, at least about 1 .0 wt% analyte per gram of chromatographic media, or any other wt% in a range bounded by any of these values. A suitable means for determining the amount of adsorption can be by a constant volume variable pressure analysis. In one embodiment, the chromatographic media may adsorb about 4.2 mg of DIPP, and/or DBABA) per gram of chromatographic media.

[0074] Without wishing to be bound by theory, it is believed that the observed retention properties of a chromatographic media can be attributed to the polarity of the oxygen- containing functional groups, such as those of graphene oxide. The polarity caused by the oxygen-containing functional groups allows the graphene oxide to form temporary chemical interaction, e.g., non-covalent interactions like ionic interaction, hydrogen bonds, Van der Waals forces with the analyte, and in doing so, retains those polar compounds more than the non-polar compounds in the sample. As a result, the chromatographic media is able to partially retain those polar analytes and separate them from the solution.

[0075] In some embodiments, the chromatographic media may separate the organic materials while not chemically transforming the organic materials. In some embodiments, the organic materials are not catalytically transformed by the chromatographic media. In some embodiments, the chromatographic media may be chemically inert relative to the analyte[s]. FIG. 1 depicts one embodiment of a method M10 for separating at least one organic material from another in a mixture. This particular embodiment of the method comprises providing a stationary phase comprising a modified graphene oxide S10, contacting a mobile phase with a modified graphene to partially retain at least one organic material with the stationary phase S20, and passing an organic eluent over the modified graphene to elute the organic materials in the mixture at different rates S30. In some embodiments, the method may further comprise selectively collecting the at least one organic material from the passed mobile phase, separating the at least one organic material from another. [0076] In some embodiments, as shown in FIGS. 3A and 3B, a stationary phase 12 comprising a modified graphene may interact with or partially retain each of two organic compounds at different retention rates, by separating the first compound 14 from the second compound 16 a distance d by the passage of an eluent as indicated by the arrow 26. Factors determining the distance d include, but are not limited to, the temperature, elution solvent eluting strength, the compound's partition /retention strength. As shown in FIG. 3B, the passage of an eluent as indicated by the arrow 26 over and past the stationary phase 12 moves the second compound 16 a distance d1 , which is greater distance than the first compound 14 having moved a distance d2. A measure of the passage of the first compound relative to the second compound can be expressed in column volumes (CV) of the eluent having passed through the reservoir as a measure of the relative distance moved from the starting position (CV) of the eluent having passed through the reservoir, e.g., d2/d1 .

[0077] In some embodiments, as shown in FIGS. 4A and 4B, a stationary phase 12 comprising a modified graphene may also have a reservoir 30 defining or having an inlet 32 and outlet 34. The embodiment may also have a dispersion 36 comprising the modified graphene with the dispersion 36 being disposed in the reservoir 30, for example, in column chromatography. As described with reference to FIGS. 3A and 3B, the stationary phase 12 interacts or partially retains each of the at least two organic compounds at different retention factors, separating the first compound 14 from the second compound 16 a distance d with the passage of an eluent as indicated by the arrow 26. Factors determining the distance d include, but are not limited to, the temperature, elution solvent eluting strength, the compound's partition/retention strength. As shown in FIG. 4B, the passage of an eluent as indicated by the arrow 26 over and through the reservoir moves the second compound 16 a distance d1 , which is greater than first compound 14, having moved a distance d2. A measure of the passage of one relative the other can be expressed in column volumes (CV) of the eluent having passed through the reservoir.

[0078] In some embodiments, as shown in FIG. 5A, a stationary phase 12 comprises a modified graphene a stationary phase 12 comprising a substrate 38 herein, and affixing the modified graphene 12 upon the substrate, and disposing a first end of the substrate in an eluent. The stationary phase 12 interacts or partially retains with each of the at least two organic compounds at different retention factors, separating the first compound 14 from the second compound 16 a distance d as the eluent travels from the first end of the substrate 40 to the second end of the substrate 42. Factors determining the distance d include, but are not limited to, the temperature, elution solvent eluting strength, the compound's partition/retention strength. As shown in FIG. 5B, the passage of an eluent as indicated by the arrow 26 over and/or through the reservoir moves the second compound 16 a distance d1 , which is greater than the first compound 14, having moved a distance d2. A measure of the passage of one relative the other can be expressed in retention factors (R_f) a distance relative to one another.

[0079] In some embodiments, the stationary phase may be free, e.g., not covalently bound or otherwise immobilized to a substrate. In some embodiments, the free stationary phase may be a plurality of beads and/or particles. In some embodiments, the stationary phase may be immobilized on support materials, particles or elements, e.g., by in situ polymerization after coating. In some embodiments, the free or immobilized stationary phase may comprise a modified graphene.

[0080] In some embodiments, the eluent may comprise a solvent, e.g., for a normal phase embodiment. In some embodiments, the eluent may comprise an organic solvent. In some embodiments, the organic solvent may include, but is not limited to, hexane, pentane, cyclohexane, benzene, dichloromethane (DCM), chloroform ether, ethyl acetate, dimethyl formamide (DMF) acetone, ethanol, methanol, tetrahydrofuran (THF), toluene, acetonitrile, diethyl ether, mixtures thereof, and/or materials having the equivalent eluting power

[0081] In some embodiments, the organic eluent comprises hexane. In some embodiments the organic eluent comprises ethyl acetate. In some embodiments, the organic eluent comprises a mixture of hexane and ethyl acetate. In some embodiments, the vol% of hexane to ethyl acetate may be from about 50 vol% to about 99 vol% hexane to about 1 vol% to about 50 vol% ethyl acetate. In one embodiment, vol% of hexane to ethyl acetate may be from about 75 vol% to about 99 vol% hexane to about 1 vol% to about 25 vol% ethyl acetate. In some embodiments, the vol% of hexane to ethyl acetate may be about 90 vol% hexane to about 10 vol% ethyl acetate.

[0082] In some embodiments, the solvent may be a non-polar solvent. In some embodiments, the solvent can be a polar solvent, e.g., for a reversed-phase embodiment. In some embodiments, the polar solvent can comprise acetonitrile, methanol, dimethyl formamide (DMF), water, or combinations thereof. In some embodiments, the polar solvent may be water. In some reversed-phase embodiments, the mobile phase may be more polar than the stationary phase. In some embodiments, the solvent may be characterized by the elements or mixture substituents having a minimum level of ability to move with or be partitioned into the mobile phase as opposed to adsorbing to the modified graphene. In some embodiments, the solvent may have an eluting power less than water.

[0083] In some embodiments, a chromatography gel for transitory retention of a material thereon is provided, the gel comprising a modified graphene. In some embodiments, a chromatography column is provided with the chromatography media or gel described herein. In some embodiments, the chromatography media, gel and/or graphene oxide may be disposed within a chromatographic column. A suitable chromatography column may be the 5 g REDISEP^® empty solid load cartridge column (Teledyne Isco, Lincoln, NE), which may be used with the COMBIFLASH^® R_F 200 flash chromatography system (Teledyne Isco, Lincoln, NE).

[0084] In some embodiments, a chromatography system is provided, the system comprising the chromatography column described herein. A suitable chromatography system can be the COMBIFLASH^® R_F 200 flash chromatography system (Teledyne Isco, Lincoln, NE).

[0085] The following are embodiments:

Embodiment 1. A method of separating at least one organic material from another in a mixture, comprising:

(a) providing a stationary phase comprising a modified graphene;

(b) contacting the mixture with the stationary phase to partially retain at least one organic material with the stationary phase; and (c) passing a mobile phase comprising an eluent through the modified graphene to elute the organic materials in the mixture at different rates.

Embodiment 2. The method of embodiment 1 , wherein providing a stationary phase comprises providing a reservoir, having an inlet and outlet defined therein, creating a dispersion comprising the modified graphene, and disposing the dispersion in the reservoir.

Embodiment 3. The method of embodiment 1 , wherein providing a stationary phase comprises providing a substrate having a surface and disposing the modified graphene on the surface.

Embodiment 4. The method of embodiment 1 , further comprising selectively collecting the at least one organic material from the passed mobile phase, separating the at least one organic material from another.

Embodiment s. The method of embodiment 1 , wherein the modified graphene comprises a graphene oxide.

Embodiment s. The method of embodiment 1 , wherein the functionalized graphene oxide is selected from reduced graphene oxide, graphene oxide, and graphene gel.

Embodiment 7. The method of embodiment 6, wherein the functionalized graphene oxide is selected from;

, and

COO.H COOH

Embodiment 8. The method of embodiment 6, wherein the graphene gel characterized by the formula:

Embodiment 9. The method of embodiment 1 , wherein the eluent comprises a non-polar solvent.

Embodiment 10. The method of embodiment 9, wherein the eluent comprises hexane.

Embodiment 11. The method of embodiment 1 , wherein the eluent comprises a polar solvent.

Embodiment 12. The method of embodiment 1 1 , wherein the eluent is selected from water, acetonitrile, methanol or DMF.

Embodiment 13. A chromatography media for transitory retention of a material thereon, comprising a gel comprising a modified graphene.

Embodiment 14. A chromatography column comprising the chromatography media of Embodiment 13. Embodiment 15. A chromatography system comprising the chromatography column of embodiment 14.

[0086] The following embodiments are contemplated:

Embodiment 1A. A method of separating organic compounds, comprising: passing a mobile phase comprising a mixture of organic compounds through a stationary phase comprising a reduced graphene oxide, a graphene oxide, a graphene gel, or a combination thereof.

Embodiment 2A. The method of embodiment 1A, wherein the reduced graphene oxide, the graphene oxide, the graphene gel, or the combination thereof is at least about 90% of the weight of the active portion of the stationary phase.

Embodiment 3A. The method of embodiment 1A or 2A, wherein the stationary phase contains less than 1 % of gold or nickel by weight.

Embodiment 4A. The method of embodiment 1A, 2A, or 3A, further comprising providing a reservoir, having an inlet and outlet, creating a dispersion comprising the stationary phase, and disposing the dispersion in the reservoir.

Embodiment 5A. The method of embodiment 1A, 2A, 3A, or 4A, further comprising disposing the reduced graphene oxide, the graphene oxide, the graphene gel, or the combination thereof on a surface of a substrate.

Embodiment 6A. The method of embodiment 1A, 2A, 3A, 4A, or 5A, wherein the method is used to separate a first organic compound having a molecular weight of about 1000 Da or less from a second organic compound having a molecular weight of about 1000 Da or less.

Embodiment 7A. The method of embodiment 1A, 2A, 3A, 4A, 5A, or 6A, further comprising selectively collecting a first organic compound from the mobile phase that has passed through the stationary phase to separate the first organic compound from a second organic compound.

Embodiment 8A. The method of embodiment 1A, 2A, 3A, 4A, 5A, 6A, or 7A, wherein the stationary phase comprises a graphene oxide.

Embodiment 9A. The method of embodiment 1A, 2A, 3A, 4A, 5A, 6A, 7A, or 8A, wherein the mobile phase comprises a non-polar solvent. Embodiment 10A. The method of embodiment 9A, wherein the mobile phase comprises hexane.

Embodiment 11 A. The method of embodiment 1A, 2A, 3A, 4A, 5A, 6A, 7A, or 8A, wherein the mobile phase comprises a polar solvent.

Embodiment 12A. The method of embodiment 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, or 1 1A, wherein the mobile phase comprises water, acetonitrile, methanol or dimethylformamide.

Embodiment 13A. A chromatography media for transitory retention of a material thereon, comprising a gel comprising a reduced graphene oxide, a graphene oxide, or a combination thereof.

Embodiment 14A. A chromatography column comprising the chromatography media of embodiment 13A.

Embodiment 15A. A chromatography system comprising the chromatography column of embodiment 14A.

EXAMPLES

[0087] It has been discovered that embodiments of elements and method described herein improve the ability of materials to be separated and identified from one another in a mixture. These benefits are further shown by the following examples, which are intended to be illustrative of the embodiments of the disclosure, but are not intended to limit the scope or underlying principles in any way.

Example 1 - Chemical retention by Reduced graphene oxide Flash Chromatography

[0088] A 5 g REDISEP^® empty solid load cartridge column (Teledyne Isco, Lincoln, NE) was packed with 4 g of reduced graphene oxide (rGO) (Graphenea, Gipuzkoa, Spain, 260-295 nm particle size, 422.69 to 499.85 m²/g BET surface area). The column was then inserted into an automated flash chromatography system (COMBIFLASH® R_F 200, Teledyne Isco, Lincoln, NE). The rGO was equilibrated with about 10-20 CV of the starting gradient of the eluent system (i.e., 9 parts hexane: 1 part ethyl acetate), at a constant flow rate of about 10 mL/min. [0089] 42 μΙ_ of the 2,6-Diisopropylphenol (DIPP) (Sigma Aldrich, St. Louis, MO, >98% PIPP) and 40 μΙ_ of the 4-(Dibutylamino)benzaldehyde (DBABA) (Sigma Aldrich, St. Louis, MO, >98% PIPP) sample mixture was dried over 0.5 g of silica for about 2 h, and then loaded directly onto the packed rGO column as described in above. The eluent flow rate was kept constant at about 4.0 mL/min and elution monitored via UV wavelength 1 (red) set at 254 nm and wavelength 2 (purple) set at 280 nm. Fractions were collected periodically by a fraction collector and the peaks were identified through thin layer chromatography (TLC) with a reference material.

[0090] The resulting chromatogram is shown as FIG. 6. The peaks are identified by the column volume (CV) indicator (marked above each fraction). The peak at about 4 CV was DIPP, while the peaks at about 12 and 17 CVs were both DBABA (cross referenced/confirmed by TLC). Both products were adequate in their respective purities (greater than 85% purity).

Example 2 - Higher Loading Ratios

[0091] A column and test sample were similarly prepared to that described in Example 1 , except that 420 μί of the DIPP and 400 μί of the DBABA sample mixture were loaded directly onto the packed rGO column

[0092] The resulting chromatogram is shown as FIG. 7. The peaks are identified by the column volumes (CV) indicator (marked above each fraction). The peak at about 4 CV was DIPP, while the peak in between 12 and 13 CV and 16 was DBABA (cross referenced by TLC). Both products were adequate in their respective purities.

Example 3 - Better Separation of Closely Running Compounds

[0093] A test column was prepared in a manner similar to that described above, except that the rGO was equilibrated with about 10-20 CV of the starting gradient of the eluent system (i.e., 1 part Ethyl Acetate: 1 part DMF), at a constant flow rate of 10 mL/min.

[0094] The sample was chromatographed in the same manner described in Example 1 , except that 40 mg of compound A ((E)-3-(4-(azepan-1 -yl)-2-fluorophenyl)acrylonitrile, Sigma-Aldrich, St. Louis, MO) and 40 mg of compound B (2-(4-(azepan-1 -yl)-2- (thfluoromethyl)benzylidene)malononitrile, Sigma-Aldrich, St. Louis, MO) sample mixture was dried over 0.5 g of silica and loaded onto the packed rGO column.

[0095] The resulting chromatogram is shown as FIG. 8. The peaks are identified by the column volumes (CV) indicator (marked above each fraction). The peak at about 8 CV was compound B, while the peak at about in between 13 and 14 CV was compound A (cross referenced by TLC) with trace amounts of B. Fraction 7 (compound B) was adequate in its purity, e.g., greater than 85% pure.

Example 4- Reusability

[0096] After running multiple separations, a column prepared as described in Example 1 , was unpacked of rGO. The rGO were washed by stirring it with 300 mL of methanol for 5 min and subsequently filtered then put into 500 mL of 0.1 M NaOH. The solution was stirred for about 5 min and then filtered. The collected rGO was then washed with 300 mL of water and filtered. The rGO was then put in 500 mL of 0.1 M HCI. After about 5 min of stirring, the rGO was filtered, and washed with 300 mL of water. The rGO was then given a final rinse with 300 mL of methanol followed by 300 mL ethyl acetate and filtered. The filtered material was stirred in 500 mL of DMF & filtered. It was washed with 500 mL DMSO and filtered. The rGO was washed again with 500 mL of methanol and filtered. A final organic wash was with 1 L of DCM before the final filtration. The collected rGO was dried and re-packed into the REDISEP® solid load cartridge column and a sample loaded and eluted as described in Example 1 above.

[0097] The resulting chromatogram is shown as FIG. 9. The peaks are identified by the column volumes (CV) indicator (marked above each fraction). The peak at about 3 CV was DIPP, while the peak at about 12 and 16 CV were both DBABA (cross referenced by TLC). Both products were adequate in their respective purities and the chromatogram matched the previous runs.

Example 5 - Reversed Phase Chromatography-A [0098] A 5 g REDISEP empty solid load cartridge column (Teledyne Isco, Lincoln, NE) was packed with 4 g of reduced graphene oxide (rGO) (Graphenea, Gipuzkoa, Spain, 260-295 nm particle size, 422.69 to 499.85 m²/g BET surface area), and was inserted into an automated flash chromatography system (COMBIFLASH^® R_F 200, Teledyne Isco, Lincoln, NE). The rGO was equilibrated with about 10-20 CV of the starting gradient of the eluent system (i.e., 7 parts water: 3 parts acetonitrile), at a constant flow rate of 25 mL/min.

[0099] A 50 mg of sodium 4-((2,3-dihydrothieno[3,4-b][1 ,4]dioxin-2-yl)oxy)butane-1 - sulfonate (EDOT-04SNa) and 50mg of methylparaben (MP) sample mixture was loaded directly onto the packed rGO column. The eluent flow rate was kept constant at 25 mL/min and elution monitored via UV wavelength 1 (red) set at 254 nm and wavelength 2 (purple) set at 280 nm. Fractions were collected periodically by a fraction collector and the peaks were identified through TLC with a reference material.

[0100] The resulting chromatogram is shown as FIG. 10. The peaks are identified by the column volume (CV) indicator (marked above each fraction). The peak at about 2 CV was EDOT-04SNa, while the peaks at about 7 CV was MP (cross referenced by TLC). Both products were adequate in their respective purities.

Example 6- Reverse Phase Chromatography-B

[0101] A 5 gm REDISEP^® empty solid load cartridge column (Teledyne Isco, Lincoln, NE) was packed with 4 gm of reduced graphene oxide (rGO) (Graphenea, Gipuzkoa, Spain, 260-295 nm particle size, 422.69 to 499.85 m²/g BET surface area], was inserted into an automated flash chromatography system (COMBIFLASH^® R_F 200, Teledyne Isco, Lincoln, NE). The rGO was equilibrated with about 10-20 CV of the starting gradient of the eluent system (i.e., 7 parts water: 3 parts acetonitrile), at a constant flow rate of 25 mL/min.

[0102] A 50 mg of the EDOT-04SNA and 50 mg of the Rhodamine B (Rh. B) sample mixture was loaded directly onto the packed rGO column. The eluent flow rate was kept constant at 25 mL/min and elution monitored via UV wavelength 1 (red) set at 254 nm and wavelength 2 (purple) set at 280 nm. Fractions were collected periodically by a fraction collector and the peaks were identified through TLC with a reference material.

[0103] The resulting chromatogram is shown as FIG. 1 1 . The peaks are identified by the column volume (CV) indicator (marked above each fraction). The peak at about 2 CV was EDOT-04SNa, while the peak at in between 5 and 6 CV was Rh. B (cross referenced by TLC). Both products were adequate in their respective purities.

[0104] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0105] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of any claim. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0106] Groupings of alternative elements or embodiments disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0107] Certain embodiments are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, the claims include all modifications and equivalents of the subject matter recited in the claims as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is contemplated unless otherwise indicated herein or otherwise clearly contradicted by context.

[0108] In closing, it is to be understood that the embodiments disclosed herein are illustrative of the principles of the claims. Other modifications that may be employed are within the scope of the claims. Thus, by way of example, but not of limitation, alternative embodiments may be utilized in accordance with the teachings herein. Accordingly, the claims are not limited to embodiments precisely as shown and described.

Claims

WHAT IS CLAIMED IS:

1 . A method of separating organic compounds, comprising: passing a mobile phase comprising a mixture of organic compounds through a stationary phase comprising a reduced graphene oxide, a graphene oxide, a graphene gel, or a combination thereof.

2. The method of claim 1 , wherein the reduced graphene oxide, the graphene oxide, the graphene gel, or the combination thereof is at least about 90% of the weight of the active portion of the stationary phase.

3. The method of claim 1 , wherein the stationary phase contains less than 1 % of gold or nickel by weight.

4. The method of claim 1 , further comprising providing a reservoir, having an inlet and outlet, creating a dispersion comprising the stationary phase, and disposing the dispersion in the reservoir.

5. The method of claim 1 , further comprising disposing the reduced graphene oxide, the graphene oxide, the graphene gel, or the combination thereof on a surface of a substrate.

6. The method of claim 1 , wherein the method is used to separate a first organic compound having a molecular weight of about 1000 Da or less from a second organic compound having a molecular weight of about 1000 Da or less.

7. The method of claim 1 , further comprising selectively collecting a first organic compound from the mobile phase that has passed through the stationary phase to separate the first organic compound from a second organic compound.

8. The method of claim 1 , wherein the stationary phase comprises a graphene oxide.

9. The method of claim 1 , wherein the mobile phase comprises a non-polar solvent.

10. The method of claim 9, wherein the mobile phase comprises hexane.

1 1 . The method of claim 1 , wherein the mobile phase comprises a polar solvent.

12. The method of claim 1 1 , wherein the mobile phase comprises water, acetonitrile, methanol or dimethylformamide.

13. A chromatography media for transitory retention of a material thereon, comprising a gel comprising a reduced graphene oxide, a graphene oxide, or a combination thereof.

14. A chromatography column comprising the chromatography media of claim 13.

15. A chromatography system comprising the chromatography column of claim 14.