Chromatographic separation of chiral biological and man-made nanostructures on a chiral stationary phase

Abstract

A stationary phase for a chiral chromatography device for separating and / or analyzing one or more target analytes is provided. The stationary phase comprises a porous active layer comprising a plurality of nanofibers, for example, comprising aluminum oxide (Al2O3), and a plurality of chiral particles, for example, comprising an assembly of nanoribbons that defines a bowtie shape. A chromatography device for separating and / or analyzing chirality of one or more target analytes in a sample by using such a stationary phase in a flow path of a chromatographic device is also provided. Further, methods of separating and / or analyzing chirality of one or more target analytes in a chiral chromatography device incorporating such a chromatographic device are also provided.

Description

CHROMATOGRAPHIC SEPARATION OF CHIRAL BIOLOGICAL AND MAN-MADE NANOSTRUCTURES ON A CHIRAL STATIONARY PHASEGOVERNMENT SUPPORT

[0001] This invention was made with government support under 1538180 awarded by the National Science Foundation. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 540,224, filed on September 25, 2023. The entire disclosure of the above application is incorporated herein by reference.FIELD

[0003] The present disclosure relates to a stationary phase for a chiral chromatography device for separating and / or analyzing one or more target analytes, a chromatography device incorporating such a stationary phase, and a method of separating and / or analyzing chirality of one or more target analytes in a chiral chromatography device.BACKGROUND

[0004] This section provides background information related to the present disclosure which is not necessarily prior art.

[0005] Chiral nanostructures possess molecular and nanoscale chirality that affords materials with unique optical, electronic, and chemical properties. Chiral nanostructures have been explored for applications in chiral sensing, enantioselective catalysis, photonics, drug delivery, and separation, among others due to the exceptionally high chiroptical activity. In view of the widespread use of chiral nanomaterials, new techniques for identifying, understanding enantio- purity and recyclability of these chiral nanostructures would be desirable. Chiral nanomaterials that simultaneously display multiscale chirality are fundamentally different from binary chirality found in traditional chiral compounds. These fundamental distinctions of multiscale chiral particles necessitate development of new types of chromatographic technologies and other chiral chromatographic separation methods. The separation process may require the separation ofproducts of synthesis of various chiral nanomaterials, including chiral pillars, chiral spiky particles, complex assemblies for gene delivery, and chiral nanoscale drugs, by way of example.

[0006] Chiral chromatography is a technique used to separate enantiomers using a chiral stationary phase (CSP). CSPs are typically a porous solid (e.g., formed of a material, like glass) coated with chiral molecules. The CSP can be packed into a column as a stationary phase for liquid chromatography (LC) or applied to a solid support (glass, aluminum, or plastic) for thin layer chromatography (TLC) separations. Common CSPs used for chromatography include polysaccharide, ligand exchange, protein, helical polymers, macrocyclic glycopeptides, and polymer brushes. From these, polysaccharides and macrocyclic glycopeptides are widely used for analytical and preparative enantioseparation. Polysaccharide base CSPs have wide use in liquidchromatography mass spectrometry for their high separation efficiency, selectivity, sensitivity and reducibly under normal and reversed-phase conditions. Macrocyclic glycopeptide antibiotics may also be capable of resolving a vast array of chiral analytes. However, there are associated drawbacks with using macrocyclic glycopeptides as CSP, including having more limited availability and potential instability, for example after exposure to extreme pH or temperature, or when exposed to certain solvent that reduces separation resolution for such CSPs.

[0007] Nanoparticles with enantiomeric recognition capabilities may make favorable candidates for stereoselective separation with enhanced resolution. Examples of nanoparticles used for chiral chromatography are gold, silver, iron oxide, and carbon based nanomaterials. The size of nanoparticles within stationary phases vary between 2 nm to a few hundred nms. These nanoparticles are surface functionalized with chiral ligands limiting their separation to molecules with molecular or surface chirality. These CSPs are limited in not being able to separate larger, rigid chiral molecules, including macromolecules, natural products, polymers, and chiral nanoparticles. Moreover, like other CSPs, solvent exposure due to solubility in certain solvents and pH can degrade such nanoparticles.

[0008] Traditional CSPs are silica particles coated with chiral molecules at the surface. Silica particles have a high surface area, which is largely contained within the internal pore structure, which increases retention and resolution of separation. Columns with pore diameters around approximately 10 nm are traditionally used for chiral analysis of pharmaceuticals and other conventional small molecules. Typically, small molecules and drugs have a molecular weight of less than 1000 Dalton (Da) with a size on the order of 1 nm. The column pore diameter is typically targeted to be three times the hydrodynamic diameter of the analyte to penetrate deep into the internal pore structure. Thus, if the pore diameter is 10 nm for a CSP, the analyte should be below 3,000 Da. However, peptides and proteins typically have a molecular weight above 10,000 Da, sofor these samples a particle with 30 nm pores should be used. There are limited options for chiral high performance liquid chromatography (HPLC) columns with pore sizes above 30 nm making separation of intact large molecules (>100 kDa) quite difficult.

[0009] Thus, chiral columns are not currently manufactured to separate large proteins and nanostructures. It would be advantageous to increase resolution to scale and apply chiral chromatographic methods to other chiral nanostructures with high molecular weights. To this end, developing new stationary phases having larger pores capable of separating a broader range of chiral nanostructures would be desirable.SUMMARY

[0010] This section provides a general summary of the disclosure and is not a comprehensive disclosure of its full scope or all of its features.

[0011] In certain aspects, the present disclosure relates to a stationary phase for a chiral chromatography device for separating and / or analyzing one or more target analytes. In certain aspects, the stationary phase comprises a porous active layer that comprises a plurality of nanofibers and a plurality of chiral particles distributed in a matrix.

[0012] In one aspect, the plurality of nanofibers comprises a material selected from the group consisting of: aluminum oxide (AI2O3), carbon, titanium dioxide (TiCh), zinc oxide (ZnO), iron oxide (Fe2O3), silicon dioxide (SiCh), zirconium silicate (ZrSiC ), germanium (Ge), silver (Si), gold (Au), copper (Cu), or any combinations thereof.

[0013] In one aspect, the plurality of nanofibers comprises aluminum oxide (AI2O3).

[0014] In one aspect, the porous active layer is disposed on a support.

[0015] In one aspect, the support comprises a material selected from the group consisting of: glass, silicon dioxide, aluminum dioxide, polyethylene terephthalate (PET), and combinations thereof.

[0016] In one aspect, the plurality of chiral particles is selected from the group consisting of: chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape, hedgehog- shaped particles, helices, nanorods, stars, cubes, prisms, nanotubes, nanosheets, nano wires, and combinations thereof.

[0017] In one aspect, the plurality of chiral particles comprises a plurality of chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape that exhibits chirality.

[0018] In one further aspect, each nanoribbon of the assembly comprises at least two peptides interconnected by at least one cadmium ion (Cd2+).

[0019] In one further aspect, each nanoribbon comprises at least two homocystine molecules interconnected by at least one cadmium ion (Cd2+).

[0020] In one further aspect, each nanoribbon has a length of greater than or equal to about 10 nanometers to less than or equal to about 100 micrometers, a thickness of greater than or equal to about 50 nanometers to less than or equal to about 10 micrometers, and a width of greater than or equal to about 3 nanometers to less than or equal to about 20 micrometers.

[0021] In one aspect, the porous active layer defines a plurality of pores having an average diameter of greater than or equal to about 10 nanometers to less than or equal to about 200 nanometers.

[0022] In one aspect, the matrix comprises a binder material comprising calcium sulfate (CaSO4).

[0023] In one aspect, the plurality of nanofibers and the plurality of chiral particles are substantially homogeneously distributed in the porous active layer.

[0024] In one aspect, the plurality of nanofibers has an average diameter of greater than or equal to about 10 nanometers to less than or equal to about 100 micrometers and an average length of greater than or equal to about 100 nanometers to less than or equal to about 100 micrometers.

[0025] In one aspect, the plurality of chiral particles has an average length of greater than or equal to about 100 nanometers to less than or equal to about 100 micrometers, an average thickness of greater than or equal to about 500 nanometers to less than or equal to about 10 micrometers, and an average width of greater than or equal to about 50 nanometers to less than or equal to about 30 micrometers.

[0026] In one aspect, a chirality of the chiral particles is within an OPD index of - 150 to +150.

[0027] In one aspect, at least a portion of the plurality of chiral particles exhibits a multiscale chirality.

[0028] In certain other aspects, the present disclosure further relates to a chromatography device for separating and / or analyzing chirality of one or more target analytes in a sample. The chromatography device comprises an inlet configured to receive the sample. The chromatography device also has a fluid flow path in which a stationary phase is disposed and that is configured to receive the sample as a mobile phase. The stationary phase comprises a porous active layer comprising a plurality of nanofibers and a plurality of chiral particles distributed in a matrix. The chromatography device also comprises an outlet through which the sample exits. The inlet, fluid flow path, and outlet are in fluid communication with one another.

[0029] In one aspect, the chromatography device is a thin layer chromatography (TLC) device and the porous active layer is a coating disposed on a planar substrate.

[0030] In one aspect, the chromatography device is a chromatographic column wherein the stationary phase is disposed in an interior of the chromatographic column.

[0031] In one aspect, the chromatographic column is a high performance liquid chromatography (HPLC) column.

[0032] In one aspect, the plurality of nanofibers comprises a material selected from the group consisting of: aluminum oxide (AI2O3), carbon, titanium dioxide (TiC ), zinc oxide (ZnO), iron oxide (Fe2O3), silicon dioxide (SiC ), zirconium silicate (ZrSiC ), germanium (Ge), silver (Si), gold (Au), copper (Cu), or any combinations thereof.

[0033] In one aspect, the plurality of nanofibers comprises aluminum oxide (AI2O3).

[0034] In one aspect, the plurality of chiral particles is selected from the group consisting of: microparticles comprising an assembly of nanoribbons that defines a bowtie shape, hedgehogshaped particles, helices, nanorods, stars, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof.

[0035] In one aspect, the plurality of chiral particles comprises a plurality of chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape that exhibits chirality.

[0036] In one further aspect, each nanoribbon comprises at least two homocystine molecules interconnected by at least one cadmium ion (Cd2+).

[0037] In one aspect, the porous active layer defines a plurality of pores having an average diameter of greater than or equal to about 10 nanometers to less than or equal to about 200 nanometers.

[0038] In one aspect, the matrix comprises an inorganic binder material comprising calcium sulfate (CaSC ).

[0039] In one aspect, the plurality of nanofibers and the plurality of chiral particles are substantially homogeneously distributed in the porous active layer.

[0040] In one aspect, at least a portion of the plurality of chiral particles exhibits a multiscale chirality.

[0041] In certain aspects, the present disclosure also relates to a method of separating and / or analyzing chirality of one or more target analytes in a chiral chromatography device. The method comprises introducing a sample comprising at least one target analyte into an inlet of the chiral chromatography device that comprises a fluid flow path in which a stationary phase is disposed. The stationary phase comprises a porous active layer comprising a plurality ofnanofibers and a plurality of chiral particles distributed in a matrix. The method further comprises separating the at least one target analyte from the sample by passing the sample as a mobile phase through the stationary phase in the fluid flow path to determine a chirality of the at least one target analyte. Then, the sample is eluted from an outlet of the chiral chromatographic device.

[0042] In one aspect, the sample comprises a plurality of enantiomers and the separating comprises enantioseparating at least a first enantiomer from a second enantiomer, where the first enantiomer is the at least one target analyte.

[0043] In one aspect, the at least one target analyte has a molecular weight greater than or equal to about 10,000 Daltons.

[0044] In one aspect, the at least one target analyte is selected from the group consisting of: carbon-based nanostructures, metal-based nanostructures, ceramic-based nanostructures, semiconductor-based nanostructure, polymeric-based nanostructures, lipids, chromosomes, ribosomes, lysosomes, peroxisomes, endosomes, exosomes, liposomes, polysomes, micelles, vesiclesomes, mesosomes, and combinations thereof.

[0045] In one aspect, the sample further comprises a solvent selected from the group consisting of: a polar protic solvent, a polar aprotic solvent, a nonpolar solvent, a protic ionic liquid, a buffer, and combinations thereof.

[0046] In one aspect, the sample further comprises an organic modifier to increase resolution of the separating, wherein the organic modifier is selected from the group consisting of: acetic acid, trifluoroacetic acid (TFA), formic acid, trimethylamine (TEA), and combinations thereof.

[0047] In one aspect, the chiral chromatography device is a thin layer chromatography (TLC) device and the porous active layer is a coating disposed on a planar substrate.

[0048] In one aspect, the chiral chromatography device is a chromatographic column wherein the stationary phase is disposed in an interior of the chromatographic column.

[0049] In one aspect, the chromatographic column is a high performance liquid chromatography (HPLC) column.

[0050] In one aspect, the plurality of chiral particles is selected from the group consisting of: microparticles comprising an assembly of nanoribbons that defines a bowtie shape, hedgehogshaped particles, helices, nanorods, stars, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof.

[0051] In one aspect, the plurality of chiral particles comprises a plurality of chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape that exhibits the chirality.

[0052] In one aspect, at least a portion of the plurality of chiral particles exhibits a multiscale chirality.

[0053] In one aspect, the plurality of nanofibers comprises a material selected from the group consisting of: aluminum oxide (AI2O3), carbon, titanium dioxide (TiC ), zinc oxide (ZnO), iron oxide (Fe2O3), silicon dioxide (SiC ), zirconium silicate (ZrSiC ), germanium (Ge), silver (Si), gold (Au), copper (Cu), or any combinations thereof.

[0054] In one aspect, the plurality of nanofibers comprises aluminum oxide (AI2O3).

[0055] In one aspect, the method further comprises after the separating, qualitatively assessing the sample.

[0056] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.DRAWINGS

[0057] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0058] FIGS. 1A-1C are scanning electron microscopy (SEM) images. FIG. 1A shows an SEM image of alumina nanofibers. FIG. IB shows an SEM image of bowtie chiral particles formed from Cd2+and L-Cystine (CST). FIG. 1C shows an SEM image of a porous active layer formed in accordance with certain aspects of the present disclosure that includes a composite of the alumina nanofibers and L-CST bowtie particles.

[0059] FIGS. 2A-2D shows characterization of chiral carbon nanoparticles (CNPs). FIG. 2A is a transmission electron microscopy (TEM) image of L-CNPs. FIG. 2B shows UV-visible spectra of chiral CNPs. FIG. 2C shows PL intensity versus wavelength showing excitation dependent emission of L-CNPs. FIG. 2D shows Fourier transform infrared spectroscopy (FT-IR) data of chiral CNPs.

[0060] FIGS. 3A-3B show an example of a chiral stationary phase thin layer chromatography (TLC) plate and CNP separation on it according to certain aspects of the present disclosure. FIG. 3A shows TLC plate having bowtie particles as chiral particles in the active layer of the stationary phase. FIG. 3B shows an example of a chiral TLC plate being used to separate CNP.

[0061] FIG. 4 shows a schematic of a chiral chromatography column using a stationary phase having alumina and L-Cystine (CST) bowtie-shaped chiral particles according to certain variations of the present disclosure.

[0062] Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings.DETAILED DESCRIPTION

[0063] Example embodiments are provided so that this disclosure will be thorough, and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0064] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of’ or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and / or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and / or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and / or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and / or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions,materials, components, elements, features, integers, operations, and / or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.

[0065] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.

[0066] When a component, element, or layer is referred to as being “on,” “engaged to,” “connected to,” or “coupled to” another element or layer, it may be directly on, engaged, connected or coupled to the other component, element, or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly engaged to,” “directly connected to,” or “directly coupled to” another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent,” etc.). As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0067] Although the terms first, second, third, etc. may be used herein to describe various steps, elements, components, regions, layers and / or sections, these steps, elements, components, regions, layers and / or sections should not be limited by these terms, unless otherwise indicated. These terms may be only used to distinguish one step, element, component, region, layer or section from another step, element, component, region, layer or section. Terms such as “first,” “second,” and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first step, element, component, region, layer or section discussed below could be termed a second step, element, component, region, layer or section without departing from the teachings of the example embodiments.

[0068] Spatially or temporally relative terms, such as “before,” “after,” “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially or temporally relative terms may be intended to encompass different orientations of the device or system in use or operation in addition to the orientation depicted in the figures.

[0069] Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working examples provided at the end of the detailed description, all numerical values ofparameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary methods of measuring and using such parameters. For example, “about” may comprise a variation of less than or equal to 5%, optionally less than or equal to 4%, optionally less than or equal to 3%, optionally less than or equal to 2%, optionally less than or equal to 1%, optionally less than or equal to 0.5%, and in certain aspects, optionally less than or equal to 0.1%.

[0070] In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges.

[0071] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0072] In certain aspects, the present disclosure contemplates a novel chiral stationary phase for chromatographic separation of chiral nanostructures. Rapid and reliable methods for identification and analysis of complex chiral mixtures is advantageous considering the wide use of chiral nanostructures in fields such as photonics, catalysis, and biomedicine. In certain aspects, the present disclosure provides a stationary phase for a chiral chromatography device that can separate, determine, and / or analyze chirality of one or more target analytes. The stationary phase comprises a porous active layer or coating that comprises both a plurality of nanofibers and a plurality of chiral particles.

[0073] The chiral particles exhibit chirality. Chirality is a geometrical property of objects or molecules that lack symmetry and cannot be superimposable onto their own mirror image. Objects with mirror images are called enantiomers and have distinct handedness: left-handed (levorotary or “L-” or “5-“) and right handed (dextrorotary or “D-” or “R-”). In certain aspects, chirality of a microparticle or nanoparticle means that the structure exhibits asymmetrical optical activity with different handedness, for example, clockwise to form left handed chirality (S- or L- orientation) and counter-clockwise to form right handed chirality (R- or D- orientation). Certain materials with microscale and / or nanoscale chirality are known to strongly rotate the polarization of linearly polarized (LinP) and circularly polarized light (CPL).

[0074] In certain variations, chiral particles may display a continuum of Osipov-Pickup- Dunmur (OPD) chirality measures that exponentially correlate with the spectral positions of thecircular dichroism peaks. In certain aspects, the chiral particles may exhibit a chirality in terms of OPD index of about -150 to about +150, optionally about -100 to about +100, optionally about -75 to about +75, optionally about -50 to about +50, optionally about -25 to about +25, optionally about -15 to about +15, optionally about -10 to about +10.

[0075] In certain variations, the chiral particles exhibit a multiscale chirality meaning a particle exhibits different chiralities at different scales and thus, the handedness can potentially be different at different scales (e.g., a single particle may be right-handed at the sub-nanometer or molecular level, but left-handed at the nanoscale, and the like). In certain aspects, this multiscale chirality enables the chiral particles to specifically capture certain molecules, like biomolecules, and make it easier to detect subtle changes in biological assemblies, such as proteins or exosomes or other extracellular vesicles.

[0076] The chiral particles may be microparticles or nanoparticles. A “microparticle” as used herein encompasses “nanoparticles,” as discussed below. In certain variations of the present teachings, a microparticle component has at least one spatial dimension that is less than about 1,000 micrometers (z.e., 1 mm), optionally less than or equal to about 100 micrometers. The term “micro-sized” or “micrometer- sized” as used herein is generally understood by those of skill in the art to mean less than about 500 micrometers (z.e., 0.5 mm). A microparticle may have at least one spatial dimension that is less than about 10 micrometers, optionally less than about 5 micrometers, optionally less than about 1 micrometer (z.e., 1,000 nm), optionally less than about 0.5 micrometers (i.e., 500 nm), and in certain aspects less than or equal to about 0.1 micrometers (z.e., 100 nm).

[0077] In certain aspects, the particles may be “nano-sized” or “nanometer-sized” and have at least one spatial dimension that is less than about 1 micrometer (i.e., 1,000 nm), optionally less than about 0.5 micrometers i.e., 500 nm), optionally less than about 0.4 micrometers (i.e., 400 nm), optionally less than about 0.3 micrometers (i.e., 300 nm), optionally less than about 0.2 micrometers (i.e., 200 nm), and in certain variations, optionally less than about 0.1 micrometers (i.e., 100 nm). In certain variations, a nanoparticle component has at least one spatial dimension that is greater than about 1 nm and less than about 1,000 nm (1 micrometer). In certain variations, a nanoparticle may have at least one spatial dimension of greater than or equal to about 1 nm to less than or equal to about 100 nm. It should be noted that so long as at least one dimension of the nanoparticle falls within the above-described nano-sized scale (for example, diameter), one or more other axes may well exceed the nano- size (for example, length and / or width).

[0078] In certain variations, the stationary phase active material comprises a plurality of axial geometry nanoparticles. Axial geometry particles are anisotropic and have a cylindrical, rod,tube, or fibrous shape with an evident elongated longitudinal axis, which is longer than the other dimensions (e.g., diameter or width), thus having an axial anisotropic geometry. Generally, an aspect ratio (AR) for cylindrical shapes (e.g., fiber, wire, pillar, rod, tube, etc.) is defined as AR = L / D, where L is the length of the longest axis (here the major longitudinal axis) and D is the diameter of the cylinder or pillar. Suitable axial geometry particles for use in the present technology generally have high aspect ratios, for example, ranging from at least about 100 to in excess of 1,000, for example. In yet other aspects, such axial geometry particles may have an aspect ratio of 5,000 or more and in certain variations 10,000 or more. The axial geometry particles will generically be referred to as “nanofibers” herein.

[0079] In certain variations, the nanofibers comprise an inorganic compound, such as a metal oxide, like alumina or aluminum oxide (AI2O3). In other alternative variations, the nanofiber may comprise carbon, titanium dioxide (TiCh), zinc oxide (ZnO), iron oxide (Fe2O3), silica or silicon dioxide (SiCh), zirconia silicon or zirconium silicate (ZrSiC ), germanium (Ge), silver (Si), gold (Au), copper (Cu), or any combinations thereof. The plurality of nanofibers may have an average diameter of less than or equal to about 100 nm, optionally greater than or equal to about 10 nm to less than or equal to about 100 nm. An average length of the nanofibers may be greater than or equal to about 100 nm. For example, the average length of the nanofiber may be greater than or equal to about 100 nm to less than or equal to about 100 micrometers.

[0080] The porous active material may be considered to be a porous composite having the nanofibers and the chiral particles distributed in a binder matrix. Thus, the porous active material may further comprise a binder material, for example, comprising calcium sulfate (CaSO4). In other variations, the binder may comprise cellulose, polymers, or derivatives thereof.

[0081] The plurality of chiral particles may be selected from the group consisting of: chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape, hedgehogshaped particles, helices, nanorods, stars, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof. For example, chiral hedgehog shaped particles may generally emulate a shape of a hedgehog animal in that the particles have needles or spikes protruding from a central core region. Thus, hedgehog- shaped particles may have a core region formed of a first material and a plurality of spikes or needles having an axial geometry, which are anisotropic with an evident elongated longitudinal axis that is longer than the other dimensions (e.g., diameter or width). The needles are connected to a surface of the core region. In various aspects, the needles are substantially orthogonal to the surface of the core region. By “substantially orthogonal,” it is meant that a longitudinal axis of the needle may form an angle with respect to the core surface(where the needle is connected) having an angle of about 90° or another angle that slightly deviates from 90°, for example, deviates up to about 15° (e.g., from about 75° to about 105°).

[0082] In certain aspects, the chiral microparticles may exhibit multiscale chirality As noted above, a particle that exhibits a multiscale chirality may exhibit different chiralities at different scales and thus, the handedness potentially can be different at different scales (e.g., a single hedgehog shaped particle may be right-handed at the sub-nanometer or molecular level, but left-handed at the nanoscale, and the like). This multiscale chirality enables the hedgehog- shaped particles to specifically capture biomolecules, and their unique interactions make it easier to detect subtle changes in biological assemblies. For example, the projections define a spiky form that creates a scaffold to capture target biomolecules or bioassemblies, such as proteins or exosomes. The multiscale chirality is also important for analyzing biological assemblies, such as exosomes or other extracellular vesicles. For example, chiral hedgehog shaped particles may generally emulate a shape of a hedgehog animal in that the particles have needles or spikes protruding from a central core region. Thus, hedgehog- shaped particles may have a core region formed of a first material and a plurality of spikes or needles having an axial geometry, which are anisotropic with an evident elongated longitudinal axis that is longer than the other dimensions (e.g., diameter or width).

[0083] The surface of the chiral particle, like the hedgehog-shaped particles, may have a biomolecules, such as an amino acid derived chiral moiety or other organic chiral moieties that serve as a shape directing moiety on the surface of the particles, provide nanoscale chirality, while the projections (e.g., spikes) provide nano or microscale chirality. For example, the chiral particle (e.g., hedgehog- shaped particle) has at least two chiralities on different scales and may have three or more distinct chiralities on three or more scales. As outlined below, the chiral particle (e.g., hedgehog- shaped particle) may exhibit distinct chirality for at least two of the scales specified, optionally for more than two of the scales specified.

[0084] By way of non-limiting example, in one variation, a first chirality of the chiral (e.g., hedgehog- shaped) particle may be on an atomic scale, for example, occurring at greater than or equal to about 1 Angstroms (0.01 nm) to less than or equal to about 10 Angstrom (0.10 nm), optionally greater than or equal to about 3 Angstroms (0.3 nm) to less than or equal to about 5 Angstrom 0.5 nm. The smallest chiral object comprises four or more atoms that do not belong to one plane corresponding to the atomic scale. 3A (Angstroms) chirality is characteristic of the a- carbon atom in organic / biomolecules (e.g., amino acid-derived moiety), such as cysteine when attached to the surface of the core region, while transition metal elements are slightly greater dimension. The chiral particle may also have a distinct second chirality on the nanoscale, forexample, at greater than or equal to about 1 nm to less than or equal to about 100 nm, and optionally about 50 nm in certain embodiments. The nanometer level of chirality is believed to be provided by the projections / spikes of a hedgehog- shaped chiral particle.

[0085] In certain further variations, the multiscale chirality further comprises a third chirality, which may be on a mesoscale. By way of example, the mesoscale chirality may be greater than or equal to about 100 nm to less than or equal to about 1,000 nm (1 micrometer). For example, the chiral particle itself may provide the third mesoscale, microscale, submillimeter, and / or millimeter scale chirality. The overall geometry of the particles can also be chiral and consistently be either left — or right-handed shapes.

[0086] The chiral particle may also have a distinct fourth chirality on the microscale, for example, at greater than or equal to about 1,000 nm (1 micrometer) to less than or equal to about 100,000 nm (100 micrometers).

[0087] The chiral particle may also have a distinct fifth chirality on the sub-millimeter scale, for example, at greater than or equal to about 10,000 nm (10 micrometers) to less than or equal to about 1,000,000 nm (1,000 micrometers or 1 mm).

[0088] Additionally, the chiral particle may also have a distinct sixth chirality on the millimeter scale, for example, at greater than or equal to about 1,000,000 nm (1 millimeter) to less than or equal to about 10,000,000 nm (10 millimeters).

[0089] In other variations, the plurality of chiral particles comprises a plurality of chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape that exhibits chirality. By bowtie shape, it is meant that a three-dimensional polyhedral structure is formed having a shape that resembles a bowtie or hourglass, for example, having a pinched or restricted central region with connected conical shapes or flares at the terminal ends. In various aspects, a chiral microparticle may comprise an assembly of a plurality of nanoribbons. The nanoribbons are assembled together and define a bowtie shape that exhibits a predetermined chirality. The chirality may be controlled by adjusting pitch, size, thickness, and length of the microparticle assembly of nanoribbons. In certain aspects, each nanoribbon of the plurality of nanoribbons comprises at least two cystine molecules interconnected by at least one cadmium ion (Cd2+). Cystine (CST) is a dipeptide of cysteine amino acid bonded via an S-S bridge, which may be interconnected by Cd2+ions. In other variations, the nanoribbons may comprise other peptides. In one alternative variation, the nanoribbon may comprise homocysteine. In certain aspects, such chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape that exhibits chirality and methods for making them are described in co-owned International Application No. PCT / US2023 / 020412 filed on April 28, 2023 entitled “Photonically Active BowtieNanoassemblies with Chirality Continuum and Applications Thereof in Machine Vision,” to Cha et al., the relevant portions of which are incorporated herein by reference.

[0090] Unlike classical chiral molecules, these bowtie- shaped particles display a continuum of Osipov-Pickup-Dunmur (OPD) chirality as noted above that may be about -150 to about +150 or any of the ranges previously specified above. Such bowtie particles may further exhibit multiscale chirality, as described above.

[0091] In certain aspects, the assembly of the bowtie microparticles can proceed by the following process. Bowties microparticles are assembled from (stacked) nanoribbons. The nanoribbons are assembled from nanoplatelets. Nanoplatelets may have an average thickness of greater than or equal to about 1 nm to less than or equal to about 2 nanometers are observed from nanoclusters, where the nanoclusters have helical molecular motifs in them. In certain variations, the microparticle assembly having a bowtie shape may have greater than 1 nanoribbon to less than or equal to about 10,000 nanoribbons.

[0092] Each respective nanoribbon may have a length of greater than or equal to about 10 nm to less than or equal to about 100 micrometers, optionally greater than or equal to about 10 nm to less than or equal to about 50 micrometers, optionally greater than or equal to about 10 nm to less than or equal to about 25 micrometers, optionally greater than or equal to about 10 nm to less than or equal to about 10 micrometers, optionally greater than or equal to about 10 nm to less than or equal to about 5 micrometers, and in certain aspects, optionally greater than or equal to about 10 nm to less than or equal to about 2 micrometers.

[0093] Each respective nanoribbon may have a thickness of greater than or equal to about 50 nm to less than or equal to about 10 micrometers, optionally greater than or equal to about 100 nm to less than or equal to about 10 micrometers, optionally greater than or equal to about 250 nm to less than or equal to about 10 micrometers, and in certain aspects, optionally greater than or equal to about 500 nm to less than or equal to about 10 micrometers.

[0094] Each respective nanoribbon may have a width of greater than or equal to about 3 nm to less than or equal to about 20 micrometers, optionally greater than or equal to about 3 nm to less than or equal to about 15 micrometers, optionally greater than or equal to about 5 nm to less than or equal to about 10 micrometers, optionally greater than or equal to about 5 nm to less than or equal to about 5 micrometers, optionally greater than or equal to about 5 nm to less than or equal to about 3 micrometers, and in certain aspects, optionally greater than or equal to about 10 nm to less than or equal to about 1 micrometer.

[0095] After the nanoribbons are assembled into the bowtie shaped microparticle, the microparticle may have a length of greater than or equal to about 100 nm to less than or equal toabout 100 micrometers, optionally greater than or equal to about 100 nm to less than or equal to about 75 micrometers, and in certain aspects, optionally greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

[0096] The bowtie shaped microparticle may have a thickness of greater than or equal to about 50 nm to less than or equal to about 10 micrometers, optionally greater than or equal to about 100 nm to less than or equal to about 10 micrometers, optionally greater than or equal to about 250 nm to less than or equal to about 10 micrometers, and in certain aspects, optionally greater than or equal to about 500 nm to less than or equal to about 10 micrometers, optionally greater than or equal to about 10 nm to less than or equal to about 5 micrometers, and in certain aspects, optionally greater than or equal to about 10 nm to less than or equal to about 2 micrometers.

[0097] The bowtie shaped microparticle may have a width of greater than or equal to about 50 nm to less than or equal to about 30 micrometers.

[0098] The bowtie shaped microparticle may have a pitch of greater than or equal to about 10 nm to less than or equal to about 100 micrometers, optionally greater than or equal to about 10 nm to less than or equal to about 50 micrometers, optionally greater than or equal to about 10 nm to less than or equal to about 5 micrometers or alternatively, optionally greater than or equal to about 200 nm to less than or equal to about 100 micrometers.

[0099] In certain aspects, methods of making the microparticles having a bowtie shape include coordinating cystine with cadmium chloride solution in the presence of water and sodium hydroxide (NaOH). Water can be replaced or supplemented with other solvents, such as ethanol, methanol, dimethyl formamide, dimethyl sulfoxide, acetonitrile, and the like. Cadmium metal serves as the bridge between cystine molecules (or peptides). In certain alternative variations, as discussed above, cystine can be replaced with longer chain homocysteine, by way of non-limiting example.

[0100] In certain aspects, chromatographic separation of chiral nanostructures on chiral nano structured microparticles is contemplated and exemplified by, but not limited to, the separation of one or more target analytes that may be chiral, such as chiral carbon nanoparticles, on a chiral stationary phase composed of standard chromatography adsorbents and chiral bowtie nano structured microparticles. Bowtie particles display chirality continuum capable of interacting with enantiomers that contain multiscale chirality.

[0101] In certain variations, the active layer (e.g., porous composite) comprising the plurality of inorganic nanofibers (e.g., alumina nanofibers) and plurality of chiral particles (e.g., bowtie shaped microparticles) and matrix is disposed on a support. The support comprises amaterial selected from the group consisting of: glass, silicon dioxide, aluminum dioxide, polymers, such as polyethylene terephthalate (PET), and combinations thereof. In certain variations, the support may be a planar or a sheet. In other variations, the support may be beads or support particles, honeycomb, other known chromatographic support shapes. In yet other variations, the stationary phase is disposed on an interior a chromatographic column of a chromatographic device, for example, as part of the packing for the chromatographic column. The packing can include beads or silica gel, alumina, polymeric resins, ion exchange resins, such as Cl 8, C8, C4 and other alkyl-bonded stationary phases. The packing materials can be poured into the column and then packed by using gentle pressure applied from a pump, for example.

[0102] Such an active layer may be porous, for example, the porous active layer may define a plurality of pores. In certain aspects, the plurality of pores may have an average diameter (D) that is at least two times the size of an average maximum dimension (d) of an analyte (D = 2 x d). By way of example, if the analyte is a nanoparticle that can have a maximum dimension of up to 100 nm, an average pore size of the pores in the stationary phase / porous active layer is at least about 200 nm or twice the size of the analyte. In certain variations, an average pore size or diameter of the porous active layer may be greater than or equal to about 10 nm to less than or equal to about 200 nm.

[0103] In certain variations, the plurality of nanofibers and the plurality of chiral particles may be present in the porous active layer at a mass ratio of about 2: 1 to 1:2, for example, at about 1:1. The plurality of nanofibers and the plurality of chiral particles may be randomly distributed within the composite forming the porous active layer or evenly or substantially homogeneously distributed in the porous active layer. The porous active layer may be a coating or thin film. In one non-limiting variation, for example, the porous active layer may have a thickness of less than or equal to about 0.1 mm, optionally greater than or equal to about 0.1 mm to less than or equal to about 0.25 mm, by way of example.

[0104] Thus, in various aspects, a chiral stationary phase for chromatography is prepared based on chiral nanostructures, for example, chiral bowties, and has been shown to be effective in analyzing samples comprising chiral carbon nanoparticles (CNPs). As will be appreciated by those of skill in the art, while chiral CNPs may be used as an example of an analyte, other chiral nanostructure materials (e.g., metals, polymers, proteins, etc.) may also be separated. The inventive technology paves the way for separating complex chiral solutions and chiral nanostructures. In various aspects, the one or more compounds or analytes to be analyzed using chromatography devices comprising the stationary phases described above may be a relatively large molecule. In certain variations, the analytes are nanoparticles, which may have an averagesize up to about 100 nm. In certain aspects, the target analyte may be greater than or equal to about 10 nm to less than or equal to about 100 nm, for example, greater than or equal to about 10 nm to less than or equal to about 75 nm greater, optionally greater than or equal to about 10 nm to less than or equal to about 50 nm, optionally greater than or equal to about 10 nm to less than or equal to about 25 nm, and in certain variations, greater than or equal to about 10 nm to less than or equal to about 20 nm. In certain other variations, the at least one target analyte may be a molecular having a molecular weight greater than or equal to about 10,000 Daltons. The at least one target analyte may be selected from the group consisting of: carbon-based nanostructures, such as chiral carbon nanoparticles, metal-based nanostructures, ceramic-based nanostructures, semiconductorbased nanostructure, polymeric -based nanostructures, lipids, chromosomes, ribosomes, lysosomes, peroxisomes, endosomes, exosomes, liposomes, polysomes, micelles, vesiclesomes, mesosomes, and combinations thereof. Separation can be achieved in both polar and non-polar solvents. Thus, in certain variations, the sample further comprises a solvent selected from the group consisting of: a polar protic solvent, a polar aprotic solvent, a nonpolar solvent, a protic ionic liquid, a buffer (e.g., a buffer for buffers for normal phase or reverse phase separation), and combinations thereof.

[0105] The present disclosure contemplates a chromatography device for separating and / or analyzing chirality of one or more target analytes in a sample. The device may comprise an inlet configured to receive the sample. The chromatography device also includes a fluid flow path in which a stationary phase is disposed and configured to receive the sample as a mobile phase. The stationary phase may be a porous active layer like any of those described above, which may include a plurality of inorganic nanofibers (e.g., comprising aluminum oxide (AI2O3)) and a plurality of chiral particles. The chromatography device also comprises an outlet through which the sample exits. Each of the inlet, fluid flow path, and outlet are in fluid communication with one another. As will be described below, in one variation, the chiral chromatography device is a thin layer chromatography (TLC) device and the porous active material is a coating disposed on a planar substrate, such as a glass substrate or slide. In other variations, the chromatography device is a chromatographic column wherein the stationary phase is disposed on an interior of the column, for example as a packed bed or packing of the chromatographic column, where the packing can include beads or silica gel, alumina, polymeric resins, ion exchange resins, such as C18, C8, C4 and other alkyl-bonded stationary phases. In such a variation, the packing materials are poured into the column and then packed by using gentle pressure applied from a pump, for example. For example, in one variation, the chromatographic column is a high performance liquid chromatography (HPLC) column.

[0106] In certain variations, the present disclosure contemplates innovative chromatographic methods for separation and analysis of chiral nanostructures using chiral particles, such as bowtie microparticles, as the chiral stationary phase (CSP) in the chromatography device. Chiral nanoparticles have been incorporated into CSPs for analysis of small molecules but not for large chiral nanostructures. Chiral nano structured microparticles incorporated into stationary phases can overcome existing challenges of chiral chromatography by offering multiple modes of separation for samples larger than about 10 to 20 nm. The following sections will discuss development of CSP using chiral microparticles, mobile phase development, and applications of novel CSP design.

[0107] The present disclosure also contemplates methods of separating and / or analyzing chirality of one or more target analytes in a sample by use of a chiral chromatography device. In one variation, such a method may comprise introducing a sample comprising at least one target analyte into an inlet of the chiral chromatography device that comprises a fluid flow path in which a stationary phase is disposed. The stationary phase comprises a porous active layer like any of those described above, which includes a plurality of nanofibers and a plurality of chiral particles. The method may comprise separating the at least one target analyte from the sample by passing the sample as a mobile phase through the stationary phase in the fluid flow path to determine a chirality of the at least one target analyte. Then the sample may be eluted from an outlet of the chiral chromatographic device. In certain aspects, the sample is a liquid sample and the chromatography is a liquid phase process.

[0108] In one aspect, the sample comprises a plurality of enantiomers and the separating comprises enantioseparating at least a first enantiomer from a second enantiomer, where the first enantiomer is the at least one target analyte. As noted above, the at least one target analyte may be a relatively large molecule, for example, having a molecular weight greater than or equal to about 10,000 Daltons. In certain aspects, the at least one target analyte may be any of those described above, including those selected from the group consisting of: carbon-based nanostructures, metal-based nanostructures, ceramic-based nanostructures, semiconductor-based nanostructure, polymeric-based nanostructures, lipids, chromosomes, ribosomes, lysosomes, peroxisomes, endosomes, exosomes, liposomes, polysomes, micelles, vesiclesomes, mesosomes, and combinations thereof. The chromatographic separation may be conducted via a normal-phase chromatography or a reverse-phase chromatography process, both of which are high performance liquid chromatography (HPLC) processes, where the polarity of the stationary phase versus mobile phase are different (either stationary phase is polar while the mobile phase is non-polar innormal phase chromatography or the stationary phase is non-polar and the mobile phase is polar in reverse phase chromatography).

[0109] Thus, the sample optionally further comprises a solvent selected from the group consisting of: a polar protic solvent, a polar aprotic solvent, a nonpolar solvent, a protic ionic liquid, a buffer, and combinations thereof. When choosing an appropriate solvent for separation, the sample may be miscible to allow migration (e.g., up the plate) but the stationary phase is not soluble in the solvent to reduce damage (e.g., to the plate) and maintain contact with the mobile phase. The sample may further comprise an organic modifier to increase resolution of the separating of the target analyte, wherein the organic modifier is selected from the group consisting of: acetic acid, trifluoroacetic acid (TFA), formic acid, trimethylamine (TEA), and combinations thereof.

[0110] As noted above, such a method may be conducted on a chiral chromatography device, such as a thin layer chromatography (TLC) device where the porous active material is a coating disposed on a planar substrate, such as a plate or a chromatographic column wherein the stationary phase is disposed within an interior of the column, such as a HPLC column.

[0111] In certain variation, the methods may further comprise qualitatively assessing the sample after the separating occurs. Qualitative assessment of the separation can be achieved after visualization using exposure to UV-light, fluorescent indicators, staining agents, or chemically treating the substrate (e.g., plate) on which the porous active layer is disposed.

[0112] Chiral CNPs were synthesized by a hydrothermal method, where 0.5 g of L- cysteine (L-cys) or D-Cysteine (D-cys) and 1 g NaOH was dissolved in 10 mL of water under ultrasonification for 20 minutes. The prepared solution was transferred into a 25 mL polytetrafluoroethylene (TEFLON®) lined autoclave and heated at 120 °C for 16 hours. The mixture was dialyzed to remove unreacted material for 3 days. The final product was filtered against a 0.22 micron filter to remove large aggregates and then lyophilized for storage.

[0113] Chiral bowtie microparticles were synthesized as follows. 10 mL stock solutions of CdCh (0.1M), L-cystine (0.1M) and NaOH (2.5 M) were prepared in de-ionized water. 1 mL of the 2.5 M NaOH solution was added to the L-cystine (L-CST) solution to solubilize. Bowties were synthesized by mixing stock solutions in a 1:1 stoichiometric ratio in aqueous media. The solution turns milky which indicates successful bowtie synthesis. The solution was allowed to sit at room temperature for 15 min to allow the reaction to run to completion. Afterwards, the bowties were spun down at 5000 rpm for 5 min 3 times with water. The bowties were freeze dried and stored in powder form prior to CSP synthesis.

[0114] Reverse Phase (RP) chiral stationary phase (CSP) Thin Layer Chromatography Plates formed in accordance with certain aspects of the present disclosure are prepared as follows. A slurry containing 250 mg of alumina nanofibers, 250 mg of bowtie particles, and 175 mg of calcium sulfate binder was mixed in 5 mL of water. The slurry was mixed and sonicated to remove large aggregates. 1.5 mL of NP- or RP-CSPs were applied to a clean microscope slide. The glass slide treated was transferred to an oven to dry at 100 °C for 30 minutes. The fabricated TLC plate was stored in vacuum until further use. To analyze samples, 10 microliters (pL) of liquid sample was applied directly to the TLC plate 5 cm above the bottom. 10 mL of mobile phase was added to a 250 mL glass beaker then the TLC plate was placed in the beaker vertically and the sample spot above the mobile phase level with the lid on top. The mobile phase was allowed to travel up the TLC for 5 minutes. The TLC plate was removed and dried at room temperature. The TLC plate was analyzed under 254 nm light excitation.

[0115] Thin Layer Chromatography (TLC) using chiral nanostructured microparticles as a stationary phase in accordance with certain aspects of the present disclosure is described as follows. Thin layer chromatography is a quick and efficient technique to separate, identify, and analyze chemical compounds. It is a planar chromatography method that uses a thin, stationary- phase coated surface to separate the components of a mixture in a sample based on different physicochemical interactions between stationary and mobile phases, also known as eluent. For certain variations of the present disclosure, alumina nanofibers shown in FIG. 1A are mixed with chiral bowties nanoassemblies shown in FIG IB to form a porous active layer that serves as the chiral stationary phase (CSP) shown in FIG. 1C, which are uniformly spread on a glass support. As discussed above, chiral bowtie assemblies are nano structured microparticles. They are hierarchically assembled from nanoribbons containing helical chains of cystine coordinated by Cd2+ ions. These bowties contain a chirality continuum capable of interacting with other materials that have multi-scale chirality.

[0116] Chiral CNP enantiomers were chosen as the samples for analysis. Chiral CNPs were synthesized from cysteine which acts as both the source of carbon and chiral precursor. To ensure the physical properties of the CNPs were similar, the reaction conditions were held constant. Because of the similarity between chiral CNPs, characterization for L-CNPs will be displayed primarily. More characterization on D-CNPs can be found in supplemental information. TEM images indicate the L-CNPs have a size distribution from 2-7 nm in FIG. 2A. In FIG. 2B, UV-visible spectra of chiral CNPs contain peaks near 200 nm and 280 nm which can be attributed to the absorption of cysteine moieties and 7t— > 7t* of sp2hybridized carbons within the crystal lattice, respectively. Excitation dependent emission of L-CNPs can be seen in FIG. 2C, withmaximum emission of 400 nm after 320 nm excitation which is typical for conjugated aromatic compounds. Fourier transform infrared spectroscopy (FT-IR) data of chiral CNPs is depicted in FIG. 2D. Peaks near 1360 - 1400 cm'1, 1600 cm'1related to COO and C=C stretching vibrations respectively. Strong adsorption bands between 3200 cm'1and 3600 cm'1can be attributed to - OH and -NH stretching vibrations. FT-IR reveals hydroxyl, carboxyl, and amine groups located at the surface of chiral CNPs.

[0117] Concentrated pots of each CNP enantiomer are applied near the bottom of the chiral TLC plate made with L-CST bowties / alumina for RP-TLC. A 250 mL beaker with a lid was used as the developing chamber. The TLC plate spotted with sample was placed vertically in the developing chamber with a small amount of water or methanol as eluent to saturate the atmosphere and equilibrate with the stationary phase. After about 5 minutes of allowing the mobile phase / sample to migrate up the column, the TLC was removed from the chamber to dry and visualized under 254 nm UV light. An example of a chiral TLC plate and CNP separation can be seen in FIGS. 3A-3B. FIG. 3 A shows the TLC plate having bowtie particles as chiral particles in the active layer of the stationary phase. FIG. 3B shows separation of chiral CNPS on such a stationary phase. The left hand spot is L-CNP and the right hand spot are D-CNP. The L-CNP migrated up the TLC plate while the D-CNPs seemed to remain as a spot.

[0118] In another variations, the stationary phase prepared in accordance with certain aspects of the present disclosure may be used in column chromatography applications. To prepare an HPLC or preparatory column, CSP is wet packed in the column by being poured into an interior of the column. Pressure may be applied within the column to remove air bubbles and to ensure adequate packing without degrading the CSP. Beads or other packing media are not necessary unless the back pressure while the HPLC is operating is too high. In this case, micrometer-sized beads are appropriate as a packing media. As noted above, these beads or packing media can be silica, polymeric, ion exchange beads, or beads coated with other chiral molecules / nanoparticles.

[0119] In this example, after charging the column with wet packed CSP, five (5) column lengths of solvent are passed over the stationary bed to remove bubbles and ensure the CSP is evenly packed. Gentle pressure from a pump is used to pack the column to ensure even packing. If the column is a preparatory column, the sample is loaded at the top of the column (inlet) and then gentle pressure is used to drive the separation and collect fractions at the top. If the column is for HPLC, the column is loaded into the LC instrument and the pump is used to pass at least 5 column lengths of solvent through the system then inject the desired sample into the LC instrument. An example of an HPLC column with Alumina / Bowtie CSP is shown in FIG. 4.

[0120] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.

Claims

CLAIMSWhat is claimed is:

1. A stationary phase for a chiral chromatography device for separating and / or analyzing one or more target analytes, the stationary phase comprising a porous active layer comprising a plurality of nanofibers and a plurality of chiral particles distributed in a matrix.

2. The stationary phase of claim 1, wherein the plurality of nanofibers comprises a material selected from the group consisting of: aluminum oxide (AI2O3), carbon, titanium dioxide (TiCh), zinc oxide (ZnO), iron oxide (Fe2O3), silicon dioxide (S 1O2), zirconium silicate (ZrSiCU), germanium (Ge), silver (Si), gold (Au), copper (Cu), or any combinations thereof.

3. The stationary phase of claim 1, wherein the plurality of nanofibers comprises aluminum oxide (AI2O3).

4. The stationary phase of claim 1, wherein the porous active layer is disposed on a support.

5. The stationary phase of claim 4, wherein the support comprises a material selected from the group consisting of: glass, silicon dioxide, aluminum dioxide, polyethylene terephthalate (PET), and combinations thereof.

6. The stationary phase of claim 1, wherein the plurality of chiral particles is selected from the group consisting of: chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape, hedgehog- shaped particles, helices, nanorods, stars, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof.

7. The stationary phase of claim 1, wherein the plurality of chiral particles comprises a plurality of chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape that exhibits chirality.

8. The stationary phase of claim 7, wherein each nanoribbon of the assembly comprises at least two peptides interconnected by at least one cadmium ion (Cd2+).

9. The stationary phase of claim 7, wherein each nanoribbon comprises at least two homocystine molecules interconnected by at least one cadmium ion (Cd2+).

10. The stationary phase of claim 7, wherein each nanoribbon has a length of greater than or equal to about 10 nanometers to less than or equal to about 100 micrometers, a thickness of greater than or equal to about 50 nanometers to less than or equal to about 10 micrometers, and a width of greater than or equal to about 3 nanometers to less than or equal to about 20 micrometers.

11. The stationary phase of claim 1, wherein the porous active layer defines a plurality of pores having an average diameter of greater than or equal to about 10 nanometers to less than or equal to about 200 nanometers.

12. The stationary phase of claim 1 wherein the matrix comprises a binder material comprising calcium sulfate (CaSC ).

13. The stationary phase of claim 1, wherein the plurality of nano fibers and the plurality of chiral particles are substantially homogeneously distributed in the porous active layer.

14. The stationary phase of claim 1, wherein the plurality of nanofibers has an average diameter of greater than or equal to about 10 nanometers to less than or equal to about 100 micrometers and an average length of greater than or equal to about 100 nanometers to less than or equal to about 100 micrometers.

15. The stationary phase of claim 1, wherein the plurality of chiral particles has an average length of greater than or equal to about 100 nanometers to less than or equal to about 100 micrometers, an average thickness of greater than or equal to about 500 nanometers to less than or equal to about 10 micrometers, and an average width of greater than or equal to about 50 nanometers to less than or equal to about 30 micrometers.

16. The stationary phase of claim 1, wherein a chirality of the chiral particles is within an OPD index of -150 to +150.

17. The stationary phase of claim 1, wherein at least a portion of the plurality of chiral particles exhibits a multiscale chirality.

18. A chromatography device for separating and / or analyzing chirality of one or more target analytes in a sample, the chromatography device comprising: an inlet configured to receive the sample; a fluid flow path in which a stationary phase is disposed and configured to receive the sample as a mobile phase, the stationary phase comprising a porous active layer comprising a plurality of nanofibers and a plurality of chiral particles distributed in a matrix; and an outlet through which the sample exits, wherein the inlet, fluid flow path, and outlet are in fluid communication with one another.

19. The chromatography device of claim 18, wherein the chromatography device is a thin layer chromatography (TLC) device and the porous active layer is a coating disposed on a planar substrate.

20. The chromatography device of claim 18, wherein the chromatography device is a chromatographic column wherein the stationary phase is disposed in an interior of the chromatographic column.

21. The chromatography device of claim 20, wherein the chromatographic column is a high performance liquid chromatography (HPLC) column.

22. The chromatography device of claim 18, wherein the plurality of nanofibers comprises a material selected from the group consisting of: aluminum oxide (AI2O3), carbon, titanium dioxide (TiCh), zinc oxide (ZnO), iron oxide (Fe2O3), silicon dioxide (SiCh), zirconium silicate (ZrSiC ), germanium (Ge), silver (Si), gold (Au), copper (Cu), or any combinations thereof.

23. The chromatography device of claim 18, wherein the plurality of nanofibers comprises aluminum oxide (AI2O3).

24. The chromatography device of claim 18, wherein the plurality of chiral particles is selected from the group consisting of: microparticles comprising an assembly of nanoribbons that defines a bowtie shape, hedgehog- shaped particles, helices, nanorods, stars, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof.

25. The chromatography device of claim 18, wherein the plurality of chiral particles comprises a plurality of chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape that exhibits chirality.

26. The chromatography device of claim 25, wherein each nanoribbon comprises at least two homocystine molecules interconnected by at least one cadmium ion (Cd2+).

27. The chromatography device of claim 18, wherein the porous active layer defines a plurality of pores having an average diameter of greater than or equal to about 10 nanometers to less than or equal to about 200 nanometers.

28. The chromatography device of claim 18, wherein the matrix comprises an inorganic binder material comprising calcium sulfate (CaSC ).

29. The chromatography device of claim 18, wherein the plurality of nanofibers and the plurality of chiral particles are substantially homogeneously distributed in the porous active layer.

30. The chromatography device of claim 18, wherein at least a portion of the plurality of chiral particles exhibits a multiscale chirality.

31. A method of separating and / or analyzing chirality of one or more target analytes in a chiral chromatography device, comprising: introducing a sample comprising at least one target analyte into an inlet of the chiral chromatography device that comprises a fluid flow path in which a stationary phase is disposed, the stationary phase comprising a porous active layer comprising a plurality of nanofibers and a plurality of chiral particles distributed in a matrix;separating the at least one target analyte from the sample by passing the sample as a mobile phase through the stationary phase in the fluid flow path to determine a chirality of the at least one target analyte; and eluting the sample from an outlet of the chiral chromatographic device.

32. The method of claim 31, wherein the sample comprises a plurality of enantiomers and the separating comprises enantioseparating at least a first enantiomer from a second enantiomer, where the first enantiomer is the at least one target analyte.

33. The method of claim 31, wherein the at least one target analyte has a molecular weight greater than or equal to about 10,000 Daltons.

34. The method of claim 31 , wherein the at least one target analyte is selected from the group consisting of: carbon-based nanostructures, metal-based nanostructures, ceramic-based nanostructures, semiconductor-based nanostructure, polymeric-based nanostructures, lipids, chromosomes, ribosomes, lysosomes, peroxisomes, endosomes, exosomes, liposomes, polysomes, micelles, vesiclesomes, mesosomes, and combinations thereof.

35. The method of claim 31, wherein the sample further comprises a solvent selected from the group consisting of: a polar protic solvent, a polar aprotic solvent, a nonpolar solvent, a protic ionic liquid, a buffer, and combinations thereof.

36. The method of claim 31 , wherein the sample further comprises an organic modifier to increase resolution of the separating, wherein the organic modifier is selected from the group consisting of: acetic acid, trifluoroacetic acid (TFA), formic acid, trimethylamine (TEA), and combinations thereof.

37. The method of claim 31, wherein the chiral chromatography device is a thin layer chromatography (TLC) device and the porous active layer is a coating disposed on a planar substrate.

38. The method of claim 31, wherein the chiral chromatography device is a chromatographic column wherein the stationary phase is disposed in an interior of the chromatographic column.

39. The method of claim 38, wherein the chromatographic column is a high performance liquid chromatography (HPLC) column.

40. The method of claim 31, wherein the plurality of chiral particles is selected from the group consisting of: microparticles comprising an assembly of nanoribbons that defines a bowtie shape, hedgehog-shaped particles, helices, nanorods, stars, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof.

41. The method of claim 31, wherein the plurality of chiral particles comprises a plurality of chiral microparticles comprising an assembly of nanoribbons that defines a bowtie shape that exhibits the chirality.

42. The method of claim 31 , wherein at least a portion of the plurality of chiral particles exhibits a multiscale chirality.

43. The method of claim 31, wherein the plurality of nanofibers comprises a material selected from the group consisting of: aluminum oxide (AI2O3), carbon, titanium dioxide (TiCh), zinc oxide (ZnO), iron oxide (Fe2O3), silicon dioxide (SiCh), zirconium silicate (ZrSiC ), germanium (Ge), silver (Si), gold (Au), copper (Cu), or any combinations thereof.

44. The method of claim 31, wherein the plurality of nanofibers comprises aluminum oxide (AI2O3).

45. The method of claim 31, further comprising after the separating, qualitatively assessing the sample.

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