Chromatographic separation of chiral bio-nanostructures and artificial nanostructures on a chiral stationary phase.

Abstract

A stationary phase is provided for a chiral chromatography apparatus for separating and / or analyzing one or more target analytes. The stationary phase comprises a porous active layer, which comprises a plurality of chiral particles, including a plurality of nanofibers, for example, aluminum oxide (Al2O3), and a plurality of nanoribbons, for example, that define a bowtie shape. A chromatography apparatus is also provided for separating and / or analyzing the chirality of one or more target analytes in a sample by using such a stationary phase in the flow path of the chromatography apparatus. Furthermore, a method for separating and / or analyzing the chirality of one or more target analytes in a chiral chromatography apparatus incorporating such a chromatography apparatus is also provided.

Description

[Technical Field]

[0001] government support This invention was made with government support under grant number 1538180 granted by the National Science Foundation. The government has certain rights in this invention.

[0002] Cross-references to related applications This application claims the benefits of U.S. Provisional Application No. 63 / 540,224, filed on 25 September 2023. The entire contents of that application are incorporated herein by reference.

[0003] This disclosure relates to a stationary phase for a chiral chromatography apparatus for separating and / or analyzing one or more target analytes, a chromatography apparatus incorporating such a stationary phase, and a method for separating and / or analyzing the chirality of one or more target analytes in a chiral chromatography apparatus. [Background technology]

[0004] This section provides background technical information related to this disclosure, but it is not necessarily prior art.

[0005] Chiral nanostructures possess chirality at both the molecular and nanoscale levels, resulting in materials with unique optical, electronic, and chemical properties. Due to their extremely high chiropractic activity, chiral nanostructures have been explored for applications including chiral sensing, enantioselective catalysis, photonics, drug delivery, and separation. Given the widespread use of chiral nanomaterials, novel techniques for identifying and understanding the enantiopurity and reusability of these chiral nanostructures are desirable. Chiral nanomaterials exhibiting simultaneous chirality at multiple scales are fundamentally different from the binary chirality observed in conventional chiral compounds. These fundamental differences in multi-scale chiral particles necessitate the development of new types of chromatography techniques and other chiral chromatographic separation methods. Separation processes may require the separation of synthetic products of various chiral nanomaterials, including, for example, chiral pillars, chiral spiky particles, complex assemblies for gene delivery, and chiral nanoscale drugs.

[0006] Chiral chromatography is a technique used to separate enantiomers using a chiral stationary phase (CSP). A CSP is typically a porous solid (e.g., formed from a material like glass) coated with chiral molecules. CSPs 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) separation. Common CSPs used in chromatography include polysaccharides, ligand exchangers, proteins, helical polymers, macrocyclic glycopeptides, and polymer brushes. Of these, polysaccharides and macrocyclic glycopeptides are widely used for analytical and conjugative enantioseparation. Polysaccharide-based CSPs are widely used in liquid chromatography-mass spectrometry due to their high separation efficiency, selectivity, sensitivity, and reproducibility under both normal-phase and reverse-phase conditions. Macrocyclic glycopeptide antibiotics can also have the ability to separate a wide range of chiral analytes. However, using macrocyclic glycopeptides as CSPs has associated drawbacks, including, for example, more limited availability and potential instability after exposure to extreme pH or temperature, or to certain solvents that reduce the separation resolution of such CSPs.

[0007] Nanoparticles with enantiomer recognition capabilities can be favorable candidates for stereoselective separation with improved separation resolution. Examples of nanoparticles used in chiral chromatography include gold, silver, iron oxide, and carbon-based nanomaterials. The size of nanoparticles in the stationary phase varies between 2 nm and several hundred nm. These nanoparticles are surface-functionalized with chiral ligands, and their separation is limited to molecules at the molecular level or those with surface chirality. These CSPs are limited in that they cannot separate larger, more rigid chiral molecules, including polymers, natural products, and chiral nanoparticles. Furthermore, as with other CSPs, solvent exposure due to solubility and pH in certain solvents can degrade such nanoparticles.

[0008] Conventional CSPs are silica particles coated with chiral molecules on their surface. Silica particles have a high surface area, primarily contained within an internal pore structure, which increases separation retention and resolution. Columns with a pore size of approximately 10 nm have traditionally been used for the chiral analysis of pharmaceuticals and other conventional small molecules. Typically, small molecules and drugs have a size of around 1 nm and a molecular weight of less than 1,000 Da. The column pore size is usually set to three times the hydrodynamic diameter of the analyte to allow deep penetration into the internal pore structure. Therefore, if the CSP pore size is 10 nm, the analyte should have a molecular weight of less than 3,000 Da. However, since peptides and proteins typically have molecular weights exceeding 10,000 Da, particles with a 30 nm pore size should be used for these samples. The selection of chiral high-performance liquid chromatography (HPLC) columns with pore sizes exceeding 30 nm is limited, making the separation of intact large molecules (≧100 kDa) extremely difficult.

[0009] Therefore, chiral columns are not currently manufactured to separate large proteins and nanostructures. It would be advantageous to increase the separation resolution on a scale and apply chiral chromatography to other chiral nanostructures with high molecular weights. For this purpose, it would be desirable to develop novel stationary phases with larger pores capable of separating a wider range of chiral nanostructures. [Overview of the project]

[0010] This section provides a general overview of the disclosure and does not constitute a comprehensive disclosure of its entire scope or all of its features.

[0011] In some embodiments, the disclosure relates to a stationary phase for a chiral chromatography apparatus for separating and / or analyzing one or more target analytes. In some embodiments, the stationary phase comprises a porous active layer containing a plurality of nanofibers and a plurality of chiral particles distributed within a matrix.

[0012] In one embodiment, the plurality of nanofibers include materials selected from the group consisting of aluminum oxide (Al2O3), carbon, titanium dioxide (TiO2), zinc oxide (ZnO), iron oxide (Fe2O3), silicon dioxide (SiO2), zirconium silicate (ZrSiO4), germanium (Ge), silver (Ag), gold (Au), copper (Cu), or any combination thereof.

[0013] In one embodiment, the nanofibers include aluminum oxide (Al2O3).

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

[0015] In one embodiment, 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 and defining a bowtie shape, hedgehog-shaped particles, spirals, nanorods, star shapes, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof.

[0017] In one aspect, the plurality of chiral particles comprises a plurality of chiral microparticles including an assembly of nanoribbons that define a bowtie shape exhibiting chirality.

[0018] In yet another aspect, each nanoribbon of the assembly comprises at least one cadmium ion (Cd 2+ ) interconnecting at least two peptides.

[0019] In yet another aspect, each nanoribbon comprises at least one cadmium ion (Cd 2+ ) interconnecting at least two homocystine molecules.

[0020] In yet another aspect, each nanoribbon has a length of from about 10 nm or more to about 100 μm or less, a thickness of from about 50 nm or more to about 10 μm or less, and a width of from about 3 nm or more to about 20 μm or less.

[0021] In one aspect, the porous active layer defines a plurality of pores having an average diameter of from about 10 nm or more to about 200 nm or less.

[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 distributed substantially uniformly within the porous active layer.

[0024] In one aspect, the plurality of nanofibers have an average diameter of from about 10 nm or more to about 100 μm or less, and an average length of from about 100 nm or more to about 100 μm or less.

[0025] In one aspect, the plurality of chiral particles have an average length of from about 100 nm or more to about 100 μm or less, an average thickness of from about 500 nm or more to about 10 μm or less, and an average width of from about 50 nm or more to about 30 μm or less.

[0026] In one aspect, the chirality of the chiral particles is within an OPD index (Osipov-Pickup-Dunmur index) of -150 to +150.

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

[0028] In several other aspects, the present disclosure further relates to a chromatography apparatus for separating and / or analyzing the chirality of one or more target analytes in a sample. The chromatography apparatus includes an inlet configured to receive the sample. The chromatography apparatus also has a fluid flow path in which a stationary phase is disposed and which 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 within a matrix. The chromatography apparatus further comprises an outlet through which the sample passes. The inlet, the fluid flow path, and the outlet are in fluid communication with one another.

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

[0030] In one aspect, the chromatography apparatus is a chromatography column, and the stationary phase is disposed inside the chromatography column.

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

[0032] In one embodiment, the plurality of nanofibers include materials selected from the group consisting of aluminum oxide (Al2O3), carbon, titanium dioxide (TiO2), zinc oxide (ZnO), iron oxide (Fe2O3), silicon dioxide (SiO2), zirconium silicate (ZrSiO4), germanium (Ge), silver (Ag), gold (Au), copper (Cu), or any combination thereof.

[0033] In one embodiment, the nanofibers include aluminum oxide (Al2O3).

[0034] In one embodiment, the multiple chiral particles are selected from the group consisting of microparticles that include aggregates of nanoribbons and define a bowtie shape, hedgehog-shaped particles, helices, nanorods, star shapes, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof.

[0035] In one embodiment, the plurality of chiral particles comprises a plurality of chiral microparticles, each containing an aggregate of nanoribbons that define a bowtie shape exhibiting chirality.

[0036] In yet another embodiment, each nanoribbon contains at least one cadmium ion (Cd 2+ It contains at least two homocystine molecules interconnected by ).

[0037] In one embodiment, the porous active layer defines a plurality of pores having an average diameter ranging from approximately 10 nm or more to approximately 200 nm or less.

[0038] In one embodiment, the matrix comprises an inorganic binder material containing calcium sulfate (CaSO4).

[0039] In one embodiment, multiple nanofibers and multiple chiral particles are substantially uniformly distributed within the porous active layer.

[0040] In one embodiment, at least one portion of a plurality of chiral particles exhibits chirality on multiple scales.

[0041] In some embodiments, the disclosure also relates to a method for separating and / or analyzing the chirality of one or more target analytes in a chiral chromatography apparatus. The method includes introducing a sample containing at least one target analyte into the inlet of a chiral chromatography apparatus having a fluid channel in which a stationary phase is disposed. The stationary phase includes a porous active layer containing a plurality of nanofibers and a plurality of chiral particles distributed within a matrix. The method further includes separating at least one target analyte from the sample in order to determine the chirality of at least one target analyte by passing the sample as a mobile phase through the stationary phase in the fluid channel. The sample is then eluted from the outlet of the chiral chromatography apparatus.

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

[0043] In one embodiment, at least one target analyte has a molecular weight of approximately 10,000 Da or more.

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

[0045] In one embodiment, the sample further comprises a solvent selected from the group consisting of polar protic solvents, polar aprotic solvents, nonpolar solvents, protic ionic liquids, buffers, and combinations thereof.

[0046] In one embodiment, the sample further comprises an organic modifier for increasing the resolution of the separation, 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 embodiment, the chiral chromatography apparatus is a thin-layer chromatography (TLC) apparatus, and the porous active layer is a coating disposed on a planar substrate.

[0048] In one embodiment, the chiral chromatography apparatus is a chromatography column, and the stationary phase is arranged inside the chromatography column.

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

[0050] In one embodiment, the multiple chiral particles are selected from the group consisting of microparticles that include aggregates of nanoribbons and define a bowtie shape, hedgehog-shaped particles, helices, nanorods, star shapes, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof.

[0051] In one embodiment, the plurality of chiral particles comprises a plurality of chiral microparticles, each containing an aggregate of nanoribbons that define a bowtie shape exhibiting chirality.

[0052] In one embodiment, at least one portion of a plurality of chiral particles exhibits chirality on multiple scales.

[0053] In one embodiment, the plurality of nanofibers include materials selected from the group consisting of aluminum oxide (Al2O3), carbon, titanium dioxide (TiO2), zinc oxide (ZnO), iron oxide (Fe2O3), silicon dioxide (SiO2), zirconium silicate (ZrSiO4), germanium (Ge), silver (Ag), gold (Au), copper (Cu), or any combination thereof.

[0054] In one embodiment, the nanofibers include aluminum oxide (Al2O3).

[0055] In one embodiment, the method further includes qualitatively evaluating the sample after separation.

[0056] Further areas of application will become apparent from the descriptions provided herein. The descriptions and specific examples in this summary are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0057] The drawings described herein are provided solely for illustrative purposes of selected embodiments and do not represent all possible embodiments, nor are they intended to limit the scope of this disclosure. [Brief explanation of the drawing]

[0058] [Figure 1] Figures 1A-1C are scanning electron microscope (SEM) images. Figure 1A is a scanning electron microscope (SEM) image of alumina nanofibers. Figure 1B is an SEM image of bowtie-shaped chiral particles formed from Cd2+ and L-cystine (CST). Figure 1C is an SEM image of a porous active layer formed according to several embodiments of this disclosure, containing a composite of alumina nanofibers and L-CST bowtie-shaped particles.

[0059] [Figure 2A]Figure 2A shows the characterization of chiral carbon nanoparticles (CNPs), specifically transmission electron microscopy (TEM) images of L-CNPs. [Figure 2B] Figure 2B shows the characterization of CNPs, specifically the ultraviolet-visible spectra of chiral CNPs. [Figure 2C] Figure 2C is a characterization of CNPs, showing the PL intensity versus wavelength, which indicates the excitation-dependent emission of L-CNPs. [Figure 2D] Figure 2D shows the characterization of CNPs, specifically Fourier transform infrared spectroscopy (FT-IR) data of chiral CNPs.

[0060] [Figure 3A] Figure 3A shows an example of a chiral stationary-phase thin-layer chromatography (TLC) plate and CNP separation thereon according to some aspects of the present disclosure, and is a TLC plate having bowtie-shaped particles as chiral particles in the active layer of the stationary phase. [Figure 3B] Figure 3B shows an example of a chiral immobilized TLC plate and CNP separation thereon according to some aspects of the present disclosure, and is a diagram showing an example of a chiral TLC plate used for separating CNPs.

[0061] [Figure 4] Figure 4 is a schematic diagram of a chiral chromatography column using a stationary phase having aluminum oxide and L-CST bowtie-shaped chiral particles, according to some modifications of the present disclosure.

[0062] Corresponding reference numerals indicate corresponding parts across multiple figures in the drawing. [Modes for carrying out the invention]

[0063] This disclosure is described in sufficient detail and provides examples so that its scope may be fully conveyed to those skilled in the art. Numerous specific details, such as examples of particular compositions, components, apparatus, and methods, are described to provide a full understanding of the embodiments of this disclosure. It will be apparent to those skilled in the art that specific details are not necessarily required, and that the exemplary embodiments may be carried out in many different forms, none of which should be construed as limiting the scope of this disclosure. In some exemplary embodiments, known processes, known apparatus structures, and known techniques are not described in detail.

[0064] The terms used herein are for the purpose of describing specific exemplary embodiments and are not intended to be limiting. The singular forms “a,” “an,” and “the” used herein may be intended to include the plural form unless the context explicitly indicates otherwise. The terms “comprises, comprising, including” and “having” are inclusive and thus identify the presence of a described feature, element, composition, step, figure, operation, and / or component, but do not exclude the presence or addition of one or more other features, figures, steps, operations, elements, components, and / or groups thereof. The open-end term “comprising” should be understood as a non-limiting term used to describe the various embodiments described herein and to define the claims; however, in some embodiments, it may be understood as a more limiting and restrictive term, such as “consisting of” or “consisting essentially of.” Accordingly, with respect to any given embodiment describing a composition, material, component, element, feature, numerical value, operation, and / or process step, the Disclosure also expressly encompasses embodiments consisting of, or substantially consisting of, the composition, material, component, element, feature, numerical value, operation, and / or process step described in this manner. In the case of "consisting of," the alternative embodiment excludes any additional composition, material, component, element, feature, numerical value, operation, and / or process step, while in the case of "consisting essentially of," any additional composition, material, component, element, feature, numerical value, operation, and / or process step that substantially affects the basic and novel properties is excluded from such embodiment, but any composition, material, component, element, feature, numerical value, operation, and / or process step that does not substantially affect the basic and novel properties may be included in that embodiment.

[0065] Any method steps, processes, and actions described herein should not be construed as necessarily requiring their execution in a specific order described or illustrated, unless such order is specified. It should also be understood that additional or alternative steps may be employed unless otherwise stated.

[0066] When a component, element, or layer is said to be “on,” “engaged to,” “connected to,” or “coupled to” another element or layer, it may be directly on, engaged to, connected to, or coupled to the other component, element, or layer, or there may be an intervening element or layer. In contrast, when an element is said to be “directly on,” “directly engaged to,” “directly connected to,” or “directly coupled to” another element or layer, there may be no intervening element or layer. Other terms used to describe relationships between elements should be interpreted by the same rules (e.g., “between” vs. “directly between,” “adjacent” vs. “directly adjacent,” etc.). As used herein, the terms "and / or" encompass all any combination of one or more of the items listed in relation to each other.

[0067] The terms "first," "second," "third," etc., may be used herein to describe various steps, elements, components, areas, layers, and / or sections, but unless otherwise stated, these steps, elements, components, areas, layers, and / or sections should not be limited by these terms. These terms may be used only to distinguish one step, element, component, area, layer, or section from other steps, elements, components, areas, layers, or sections. Where used herein, terms such as "first," "second," and other numerical terms do not imply order or sequence unless explicitly indicated by the context. Accordingly, the first step, element, component, area, layer, or section described below may be referred to as the second step, element, component, area, layer, or section without departing from the teaching of the exemplary embodiments.

[0068] Spatial or temporally relative terms such as "before," "after," "inner," "outer," "beneath," "below," "lower," "above," and "upper" may be used herein for ease of description to describe the relationship between one element or feature and another, as illustrated in the figures. Spatial or temporally relative terms may be intended to encompass different orientations of a device or system in use or operation, in addition to the orientation illustrated in the figures.

[0069] Throughout this disclosure, numerical values ​​represent slight deviations from a given value and approximate measurements or limits of range to encompass embodiments having approximately that value and embodiments having exactly that value. All numerical values ​​of parameters (e.g., quantities or conditions) in this specification, including the appended claims, except for the examples provided at the end of the detailed description, should be understood in all cases as being modified by the term "about," whether or not "about" actually appears before the numerical value. "About" indicates that the stated numerical value allows for a certain degree of slight inaccuracy (the value approaches the value with some degree of accuracy; is approximately or reasonably close to the value; is nearly the value). Where the inaccuracy provided by "about" is not understood in this ordinary sense in the art, "about" as used herein indicates at least the variation that may arise from the ordinary methods of measuring and using such parameters. For example, "about" may include fluctuations of 5% or less, arbitrarily 4% or less, arbitrarily 3% or less, arbitrarily 2% or less, arbitrarily 1% or less, arbitrarily 0.5% or less, and in certain embodiments, arbitrarily 0.1% or less.

[0070] Furthermore, the disclosure of the range includes the endpoints and subranges given for the range, as well as the disclosure of all values ​​within the entire range and any further subdivided ranges.

[0071] Exemplary embodiments are described in further detail below with reference to the accompanying diagrams.

[0072] In some embodiments, the disclosure provides novel chiral stationary phases for chromatographic separation of chiral nanostructures. Given the widespread use of chiral nanostructures in fields such as photonics, catalysis, and biomedicine, a rapid and reliable method for the identification and analysis of complex chiral mixtures is advantageous. In some embodiments, the disclosure provides stationary phases for chiral chromatography apparatus capable of separating, determining, and / or analyzing the chirality of one or more target analytes. The stationary phase comprises a porous active layer or coating containing both multiple nanofibers and multiple chiral particles.

[0073] Chiral particles exhibit chirality. Chirality is a geometric property of an object or molecule that lacks symmetry and cannot be superimposed on its own mirror image. Objects with mirror images are called enantiomers and have different orientations, namely left-handed (levorotatory or "L-" or "S-") and right-handed (dextrorotatory or "D-" or "R-"). In some embodiments, the chirality of microparticles or nanoparticles means that the structure exhibits asymmetric optical activity with different orientations, for example, forming left-handed chirality (S- or L- orientation) by clockwise rotation and right-handed chirality (R- or D- orientation) by counterclockwise rotation. Certain materials with microscale and / or nanoscale chirality are known to strongly rotate the polarization of linearly polarized (LinP) and circularly polarized (CPL) light.

[0074] In some variations, chiral particles may exhibit a continuum of Osipov-Pickup-Dunmur (OPD) chirality indices that correlate exponentially with the spectral position of the circular dichroism peak. In some embodiments, chiral particles may exhibit chirality with respect to the OPD index from approximately -150 to approximately +150, arbitrarily from approximately -100 to approximately +100, arbitrarily from approximately -75 to approximately +75, arbitrarily from approximately -50 to approximately +50, arbitrarily from approximately -25 to approximately +25, arbitrarily from approximately -15 to approximately +15, and arbitrarily from approximately -10 to approximately +10.

[0075] In some modifications, chiral particles exhibit multi-scale chirality, meaning that particles exhibit different chirality at different scales, and therefore their handedness can potentially differ at different scales (for example, a single particle may be right-handed at the sub-nanometer or molecular level, but left-handed at the nanoscale, etc.). In some embodiments, this multi-scale chirality allows chiral particles to specifically capture certain molecules, such as biomolecules, and to more easily detect subtle changes in biological aggregates such as proteins, exosomes, or other extracellular vesicles.

[0076] Chiral particles may be microparticles or nanoparticles. As used herein, “microparticles” encompasses “nanoparticles” as described below. In some modifications of the teachings of the subject, the microparticle components have at least one spatial dimension less than about 1,000 μm (i.e., 1 mm) and optionally less than or equal to about 100 μm. As used herein, the terms “microsize” or “micrometer size” are generally understood by those skilled in the art to mean less than about 500 μm (i.e., 0.5 mm). Microparticles may have at least one spatial dimension less than about 10 μm, optionally less than about 5 μm, optionally less than about 1 μm (i.e., 1,000 nm), optionally less than about 0.5 μm (i.e., 500 nm), and in some embodiments less than or equal to about 0.1 μm (i.e., 100 nm).

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

[0078] In some modifications, the stationary phase-active material comprises a plurality of axially symmetric nanoparticles. The axially symmetric particles are anisotropic and have cylindrical, rod-shaped, tubular, or fibrous shapes, with a distinct longitudinal axis longer than other dimensions (e.g., diameter or width), and thus have an axially anisotropic shape. Generally, the aspect ratio (AR) of a cylindrical shape (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 main longitudinal axis) and D is the diameter of the cylinder or pillar. Axially symmetric particles suitable for use in this art generally have high aspect ratios, for example, ranging from at least about 100 to over 1,000. In yet another embodiment, such axially symmetric particles may have aspect ratios of 5,000 or more, and in some modifications, over 10,000. In this specification, such axially symmetric particles are generally referred to as “nanofibers”.

[0079] In some variations, the nanofibers may include inorganic compounds such as metal oxides, e.g., alumina or aluminum oxide (Al2O3). In other alternative variations, the nanofibers may include carbon, titanium dioxide (TiO2), zinc oxide (ZnO), iron oxide (Fe2O3), silica or silicon dioxide (SiO2), zirconia silicon or zirconium silicate (ZrSiO4), germanium (Ge), silver (Ag), gold (Au), copper (Cu), or any combination thereof. Multiple nanofibers may have an average diameter of about 100 nm or less, and optionally between about 10 nm and about 100 nm. The average length of the nanofibers may be about 100 nm or more. For example, the average length of the nanofibers may be between about 100 nm and about 100 μm.

[0080] Porous active materials can be considered as porous composites in which nanofibers and chiral particles are distributed within a binder matrix. Therefore, porous active materials may further include a binder material, for example, calcium sulfate (CaSO4). In other variations, the binder may include cellulose, polymers, or derivatives thereof.

[0081] Multiple chiral particles can be selected from the group consisting of chiral microparticles that include aggregates of nanoribbons and define a bowtie shape, hedgehog-shaped particles, helices, nanorods, star shapes, cubes, prisms, nanotubes, nanosheets, nanowires, and combinations thereof. For example, chiral hedgehog-shaped particles can generally mimic the shape of a hedgehog in that the particle has needles or spikes protruding from a central core region. Thus, a hedgehog-shaped particle may have a core region formed from a first material, and multiple axially symmetric spikes or needles, which are anisotropic and have a distinct longitudinal axis longer than other dimensions (e.g., diameter or width). The needles are connected to the surface of the core region. In various embodiments, the needles are substantially orthogonal to the surface of the core region. "Substantially orthogonal" means that the longitudinal axis of the needles can form an angle of about 90° with respect to the core surface (where the needles are connected), or other angles that deviate slightly from 90°, for example, up to about 15° (e.g., from about 75° to about 105°).

[0082] In some embodiments, chiral microparticles can exhibit multi-scale chirality. As mentioned above, particles exhibiting multi-scale chirality can exhibit different chirality at different scales, and therefore their handedness can differ at different scales (for example, a single hedgehog-shaped particle may be right-handed at the sub-nanometer or molecular level, but left-handed at the nanoscale). This multi-scale chirality allows hedgehog-shaped particles to specifically capture biomolecules, and their unique interactions can more easily detect subtle changes in biological aggregates. For example, protrusions define spike-like morphologies that form scaffolds for capturing target biomolecules or biological aggregates such as proteins or exosomes. Multi-scale chirality is also important for analyzing biological aggregates such as exosomes or other extracellular vesicles. For example, chiral hedgehog-shaped particles can generally mimic the shape of a hedgehog in that the particle has needles or spikes protruding from a central core region. Therefore, hedgehog-shaped particles may have a core region formed from a first material, and a plurality of axially symmetrical spikes or needles, which are anisotropic and have a distinct longitudinal axis longer than other dimensions (e.g., diameter or width).

[0083] The surface of chiral particles, like hedgehog-shaped particles, may have biomolecules such as amino acid-derived chiral moieties or other organic chiral moieties, which function as shape-directing moieties on the particle surface and can provide nanoscale chirality, while protrusions (e.g., spikes) provide nanoscale or microscale chirality. For example, a chiral particle (e.g., a hedgehog-shaped particle) may have at least two chiralities at different scales and three or more different chiralities at three or more scales. As outlined below, a chiral particle (e.g., a hedgehog-shaped particle) may exhibit different chiralities at at least two of a specified scale and may exhibit different chiralities at more than two of any specified scales.

[0084] As a non-limiting example, in one variation, the first chirality of a chiral (e.g., hedgehog-shaped) particle can be at the atomic scale, for example, between about 1 angstrom (Å) (0.01 nm) and about 10 Å (0.10 nm), and optionally between about 3 Å (0.3 nm) and about 5 Å (0.5 nm). The smallest chiral object contains four or more atoms that do not belong to a single plane, which corresponds to the atomic scale. The 3 Å chirality is characteristic of α-carbon atoms in organic / biomolecules (e.g., amino acid-derived portions), and is seen, for example, when cysteine ​​is bonded to the surface of the core region, while transition metal elements have slightly larger dimensions. Chiral particles may also have a different second chirality at the nanoscale, for example between about 1 nm and about 100 nm, and optionally about 50 nm in some embodiments. Nanometer-level chirality is thought to be provided by the protrusions / spikes of the hedgehog-shaped chiral particle.

[0085] In some further variations, multiple-scale chirality may include a third chirality, which can be mesoscale. For example, mesoscale chirality may be between approximately 100 nm and approximately 1,000 nm (1 micrometer). For instance, a chiral particle itself may provide a third mesoscale, microscale, submillimeter-scale, and / or millimeter-scale chirality. The overall shape of the particle may also be chiral and consistently either left-handed or right-handed.

[0086] Chiral particles may also possess a fourth chirality that differs at the microscale, for example, between approximately 1,000 nm (1 μm) and approximately 100,000 nm (100 μm).

[0087] Chiral particles may also have a different fifth chirality on the submillimeter scale, for example, above about 10,000 nm (10 μm) and below about 1,000,000 nm (1,000 μm or 1 mm).

[0088] Furthermore, chiral particles may also possess a different sixth chirality on a millimeter scale, for example, between approximately 1,000,000 nm (1 mm) and approximately 10,000,000 nm (10 mm).

[0089] In other modifications, the multiple chiral particles include multiple chiral microparticles, each comprising an aggregate of nanoribbons defining a bowtie shape exhibiting chirality. The bowtie shape means the formation of a three-dimensional polyhedral structure having a shape similar to, for example, a bowtie or hourglass, for example, having a constricted or restricted central region with conical or flared ends. In various embodiments, the chiral microparticles may comprise aggregates of multiple nanoribbons. The nanoribbons aggregate together to define a bowtie shape exhibiting a predetermined chirality. Chirality can be controlled by adjusting the pitch, size, thickness, and length of the nanoribbon microparticle aggregate. In some embodiments, each nanoribbon of the multiple nanoribbons contains at least one cadmium ion (Cd 2+ It contains at least two cystine molecules interconnected by ) . Cystine (CST) is a dipeptide of cysteine ​​amino acids linked via SS bonds, which is Cd 2+ They can be interconnected by ions. In other modifications, the nanoribbons may contain other peptides. In one alternative modification, the nanoribbons may contain homocysteine. In some embodiments, such chiral microparticles, including aggregates of nanoribbons defining a chiral bowtie shape, and methods for producing them, are described in the jointly owned international application PCT / US2023 / 020412, filed on 28 April 2023, entitled "Photonically Active Bowtie Nanoassemblies with Chirality Continuum and Applications Thereof in Machine Vision" by Cha et al., the relevant portions of which are incorporated herein by reference.

[0090] Unlike conventional chiral molecules, these bowtie-shaped particles can exhibit a continuum of Osipov-Pickup-Dunmur (OPD) chirality, as described above, ranging from approximately -150 to approximately +150, or any of the ranges already defined above. Such bowtie particles can also exhibit chirality on multiple scales, as described above.

[0091] In some embodiments, aggregates of bowtie microparticles can be formed by the following process: Bowtie microparticles are assembled from (stacked) nanoribbons. Nanoribbons are assembled from nanoplatelets. Nanoplatelets are observed as nanoclusters, where the nanoclusters have helical molecular motifs internally and may have an average thickness of about 1 nm or more and about 2 nm or less. In some modifications, the bowtie-shaped microparticle aggregates may have nanoribbons greater than 1 and less than or equal to about 10,000.

[0092] Each nanoribbon may have a length of approximately 10 nm or more and approximately 100 μm or less, optionally approximately 10 nm or more and approximately 50 μm or less, optionally approximately 10 nm or more and approximately 25 μm or less, optionally approximately 10 nm or more and approximately 10 μm or less, optionally approximately 10 nm or more and approximately 5 μm or less, and in some embodiments, optionally approximately 10 nm or more and approximately 2 μm or less.

[0093] Each nanoribbon may have a thickness of approximately 50 nm or more and approximately 10 μm or less, optionally approximately 100 nm or more and approximately 10 μm or less, optionally approximately 250 nm or more and approximately 10 μm or less, and in some embodiments, optionally approximately 500 nm or more and approximately 10 μm or less.

[0094] Each nanoribbon may have a width of approximately 3 nm or more and approximately 20 μm or less, optionally approximately 3 nm or more and approximately 15 μm or less, optionally approximately 5 nm or more and approximately 10 μm or less, optionally approximately 5 nm or more and approximately 5 μm or less, optionally approximately 5 nm or more and approximately 3 μm or less, and in some embodiments, optionally approximately 10 nm or more and approximately 1 μm or less.

[0095] After the nanoribbons are assembled into bowtie-shaped microparticles, the microparticles may have a length of approximately 100 nm or more and approximately 100 μm or less, optionally, approximately 100 nm or more and approximately 75 μm or less, and in some embodiments, optionally, approximately 100 nm or more and approximately 50 μm or less.

[0096] The bowtie-shaped microparticles may have a thickness of approximately 50 nm or more and approximately 10 μm or less, optionally approximately 100 nm or more and approximately 10 μm or less, optionally approximately 250 nm or more and approximately 10 μm or less, and in some embodiments optionally approximately 500 nm or more and approximately 10 μm or less, optionally approximately 10 nm or more and approximately 5 μm or less, and in some embodiments optionally approximately 10 nm or more and approximately 2 μm or less.

[0097] Bowtie-shaped microparticles can have a width ranging from approximately 50 nm or more to approximately 30 μm or less.

[0098] Bowtie-shaped microparticles may have a pitch of approximately 10 nm or more and approximately 100 μm or less, optionally approximately 10 nm or more and approximately 50 μm or less, optionally approximately 10 nm or more and approximately 5 μm or less, or alternatively, optionally approximately 200 nm or more and approximately 100 μm or less.

[0099] In some embodiments, a method for producing bowtie-shaped microparticles involves coordinating cystine with a cadmium chloride solution in the presence of water and sodium hydroxide (NaOH). Water may be substituted or complemented by other solvents such as ethanol, methanol, dimethylformamide, dimethyl sulfoxide, acetonitrile, and the like. The cadmium metal acts as a bridge between cystine molecules (or peptides). In some alternative modifications, as already described, and as a non-limiting example, cystine may be substituted with longer-chain homocysteine.

[0100] In some embodiments, chromatographic separation of chiral nanostructures on chiral nanostructured microparticles is envisioned and exemplified by the separation of one or more potentially chiral target analytes, such as chiral carbon nanoparticles, where the separation is performed on a chiral stationary phase consisting of a standard chromatographic adsorbent and chiral bowtie nanostructured microparticles. The bowtie particles exhibit a chirality continuum capable of interacting with enantiomers having multiple scales of chirality.

[0101] In some variations, an active layer (e.g., a porous composite) containing multiple inorganic nanofibers (e.g., alumina nanofibers) and multiple chiral particles (e.g., bowtie-shaped microparticles) and a matrix is ​​disposed on a support. The support includes materials selected from the group consisting of glass, silicon dioxide, aluminum dioxide, polymers, e.g., polyethylene terephthalate (PET), and combinations thereof. In some variations, the support may be planar or sheet. In other variations, the support may be beads or support particles, honeycomb, or other known chromatographic support shapes. In yet another variation, the stationary phase is disposed inside the chromatographic column of a chromatograph, for example, as part of the packing of the chromatographic column. The packing may include beads or silica gel, alumina, polymer resins, ion exchange resins, e.g., C18, C8, C4, and other alkyl-bonded stationary phases. The packing may be injected into the column and then packed using gentle pressure, for example, applied from a pump.

[0102] Such an active layer may be porous, and for example, a porous active layer may define multiple pores. In some embodiments, the multiple pores may have an average diameter (D) that is at least twice the average maximum dimension (d) of the analyte (D = 2xd). For example, if the analyte is a nanoparticle that may have a maximum dimension of up to 100 nm, the 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 some variations, the average pore size or diameter of the porous active layer may range from about 10 nm to about 200 nm.

[0103] In some variations, multiple nanofibers and multiple chiral particles may be present in the porous active layer in a mass ratio of about 2:1 to 1:2, for example, about 1:1. The multiple nanofibers and multiple chiral particles may be randomly distributed within the composite forming the porous active layer, or they may be uniformly or substantially uniformly distributed within the porous active layer. The porous active layer may be a coating or a thin film. In one non-limiting variation, for example, the porous active layer may have a thickness of about 0.1 mm or less, and may be arbitrarily between about 0.1 mm and about 0.25 mm.

[0104] Therefore, in various embodiments, chiral stationary phases for chromatography have been shown to be effective when preparing chiral nanostructures, such as chiral bowties, and when analyzing samples containing chiral carbon nanoparticles (CNPs). As will be understood by those skilled in the art, while chiral CNPs can be used as an example of an analyte, other chiral nanostructure materials (e.g., metals, polymers, proteins, etc.) can also be separated. The techniques of the present invention pave the way for separating complex chiral solutions and chiral nanostructures. In various embodiments, one or more compounds or analytes analyzed using a chromatographic apparatus containing the stationary phases described above may be relatively large molecules. In some modifications, the analytes are nanoparticles and may have an average size up to about 100 nm. In some embodiments, the target analyte may be between about 10 nm and about 100 nm, for example, between about 10 nm and about 75 nm, optionally between about 10 nm and about 50 nm, optionally between about 10 nm and about 25 nm, and in some modifications, between about 10 nm and about 20 nm. In some other variations, at least one target analyte may be a molecule having a molecular weight of approximately 10,000 Da or greater. At least one target analyte may be selected from the group consisting of carbon nanostructures such as chiral carbon nanoparticles, metal nanostructures, ceramic nanostructures, semiconductor nanostructures, polymer nanostructures, lipids, chromosomes, ribosomes, lysosomes, peroxisomes, endosomes, exosomes, liposomes, polysomes, micelles, vesiclesomes, mesosomes, and combinations thereof. Separation can be achieved in both polar and nonpolar solvents. Therefore, in some variations, the sample further includes a solvent selected from the group consisting of polar protic solvents, polar aprotic solvents, nonpolar solvents, protic ionic liquids, buffers (e.g., buffers for normal-phase or reversed-phase separation), and combinations thereof.

[0105] This disclosure envisions a chromatography apparatus for separating and / or analyzing the chirality of one or more target analytes in a sample. The apparatus may include an inlet configured to receive a sample. The chromatography apparatus further includes a fluid channel configured to receive a sample as a mobile phase, with a stationary phase disposed therein. The stationary phase may be a porous active layer similar to any of those described above, and may include a plurality of inorganic nanofibers (e.g., aluminum oxide (Al2O3)) and a plurality of chiral particles. The chromatography apparatus also includes an outlet from which the sample is discharged. The inlet, fluid channel, and outlet are each in fluid communication with one another. In one modification, as described below, the chiral chromatography apparatus is a thin-layer chromatography (TLC) apparatus, and the porous active material is a coating disposed on a planar substrate such as a glass substrate or slide. In another modification, the chromatography apparatus is a chromatography column, and the stationary phase is disposed inside the column, for example as a packed bed or packing of the chromatography column, where the packing may include beads or silica gel, alumina, polymer resin, ion exchange resin, e.g., C18, C8, C4, and other alkyl-bonded stationary phases. In such modifications, the packing material is injected into the column and then packed using a gentle pressure, for example, applied from a pump. For example, in one modification, the chromatography column is a high-performance liquid chromatography (HPLC) column.

[0106] In several modifications, this disclosure provides an innovative chromatographic method for the separation and analysis of chiral nanostructures using chiral particles, such as bowtie microparticles, as a chiral stationary phase (CSP) in a chromatographic apparatus. While chiral nanoparticles have been incorporated into CSPs for the analysis of small molecules, they have not been incorporated for larger chiral nanostructures. Chiral nanostructured microparticles incorporated into the stationary phase can overcome existing challenges in chiral chromatography by providing multiple separation modes for samples larger than approximately 10 to 20 nm. The following sections describe the development of CSPs using chiral microparticles, the development of mobile phases, and the applications of novel CSP designs.

[0107] This disclosure also envisions a method for separating and / or analyzing the chirality of one or more target analytes in a sample by using a chiral chromatography apparatus. In one modification, such a method may include introducing a sample containing at least one target analyte into the inlet of a chiral chromatography apparatus having a fluid channel in which a stationary phase is disposed. The stationary phase includes a porous active layer such as any of those described above, which includes a plurality of nanofibers and a plurality of chiral particles. The method may include separating at least one target analyte from the sample and determining the chirality of at least one target analyte by passing the sample as a mobile phase through the stationary phase in the fluid channel. The sample can then be eluted from the outlet of the chiral chromatography apparatus. In some embodiments, the sample is a liquid sample and chromatography is a liquid-phase process.

[0108] In one embodiment, the sample comprises multiple enantiomers, and the separation comprises enantio-separation of at least a first enantiomer from a second enantiomer, where the first enantiomer is at least one target analyte. As described above, the at least one target analyte may be a relatively large molecule, for example, having a molecular weight of about 10,000 Da or more. In some embodiments, the at least one target analyte may be any of those described above, and is selected from the group consisting of carbon nanostructures, metal nanostructures, ceramic nanostructures, semiconductor nanostructures, polymer nanostructures, lipids, chromosomes, ribosomes, lysosomes, peroxisomes, endosomes, exosomes, liposomes, polysomes, micelles, vesiclesomes, mesosomes, and combinations thereof. Chromatographic separation can be carried out via normal-phase chromatography or reverse-phase chromatography, both of which are high-performance liquid chromatography (HPLC) processes, where the polarity of the stationary phase and the mobile phase are different (either in normal-phase chromatography the stationary phase is polar and the mobile phase is nonpolar, or in reverse-phase chromatography the stationary phase is nonpolar and the mobile phase is polar).

[0109] Therefore, the sample optionally further includes a solvent selected from the group consisting of polar protic solvents, polar aprotic solvents, nonpolar solvents, protic ionic liquids, buffers, and combinations thereof. When selecting a suitable solvent for separation, the sample may be miscible to allow movement (e.g., upward movement of the plate), while the stationary phase is not soluble in the solvent to reduce damage (e.g., damage to the plate) and to maintain contact with the mobile phase. The sample may further include an organic modifier to increase the resolution of the separation of the target analyte, where the organic modifier is selected from the group consisting of acetic acid, trifluoroacetic acid (TFA), formic acid, trimethylamine (TEA), and combinations thereof.

[0110] As described above, such methods can be carried out on a chiral chromatography apparatus, for example, a thin-layer chromatography (TLC) apparatus having a porous active material which is a coating disposed on a planar substrate such as a plate, or a chromatography column in which the stationary phase is disposed inside the column, such as an HPLC column.

[0111] In some variations, the method may further include qualitative evaluation of the sample after separation has occurred. Qualitative evaluation of the separation may be performed after visualization using exposure to ultraviolet light, a fluorescent indicator, a stain, or chemical treatment of a substrate (e.g., a plate) on which a porous active layer is provided.

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

[0113] Chiral bowtie microparticles were synthesized as follows: A 10 mL stock solution of CdCl2 (0.1 M), L-cystine (0.1 M), and NaOH (2.5 M) was prepared in deionized water. 1 mL of 2.5 M NaOH solution was added to the L-cystine (L-CST) solution for solubilization. Bowties were synthesized by mixing the stock solutions in an aqueous medium in a 1:1 stoichiometric ratio. The solution turned milky white, indicating successful bowtie synthesis. The solution was allowed to stand at room temperature for 15 minutes to allow the reaction to complete. The bowties were then centrifuged three times at 5,000 rpm for 5 minutes each using water. The bowties were lyophilized and stored in powder form prior to CSP synthesis.

[0114] Reverse-phase (RP) chiral stationary phase (CSP) thin-layer chromatography plates formed according to some aspects of this 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 was spread onto a clean microscope slide. The processed glass slide was transferred to an oven to dry at 100°C for 30 minutes. The prepared TLC plates were stored in vacuum until further use. To analyze a sample, 10 microliters (μL) of liquid sample was spread directly onto the TLC plate 5 cm above the bottom. 10 mL of mobile phase was added to a 250 mL glass beaker, and then the TLC plate was placed vertically in the beaker so that the sample spot was above the level of the mobile phase, and a lid was placed on top. The mobile phase was moved upwards on the TLC plate for 5 minutes. The TLC plate was removed and dried at room temperature. The TLC plate was analyzed under 254 nm photoexcitation.

[0115] In accordance with some aspects of this disclosure, thin-layer chromatography (TLC) using chiral nanostructured microparticles as the stationary phase is described as follows: Thin-layer chromatography is a rapid and efficient technique for separating, identifying, and analyzing chemical compounds. It is a planar chromatography method that uses a thin stationary phase coated surface to separate components of a mixture in a sample based on different physicochemical interactions between the stationary phase and the mobile phase (also known as the eluent). In some modifications of this disclosure, alumina nanofibers shown in Figure 1A are mixed with chiral bowtie nanoassemblies shown in Figure 1B and uniformly spread on a glass support to form a porous active layer that functions as a chiral stationary phase (CSP) shown in Figure 1C. As already described, the chiral bowtie assemblies are nanostructured microparticles. They are Cd 2It is hierarchically assembled from nanoribbons containing helical chains of cystine coordinated by cations. These bowties contain a chirality continuum capable of interacting with other materials having multi-scale chirality.

[0116] Chiral CNP enantiomers were selected as samples for analysis. Chiral CNPs were synthesized from cysteine, which functions as both the carbon source and the chiral precursor. To ensure that the physical properties of the CNPs are similar, the reaction conditions were kept constant. Due to the similarity between chiral CNPs, the characterization of L-CNP is mainly presented. Further characterization regarding D-CNP can be found in the supplementary information. In Figure 2A, TEM images show that L-CNP has a size distribution of 2 to 7 nm. In Figure 2B, the UV-visible spectrum of chiral CNPs includes peaks around approximately 200 nm and approximately 280 nm, which correspond to the absorption of the cysteine moiety and sp in the crystal lattice, respectively 2 hybridized carbon π→π * transition. In Figure 2C, excitation-dependent luminescence of L-CNP is observed, showing a maximum emission at 400 nm after 320 nm excitation, which is typical for conjugated aromatic compounds. The Fourier-transform infrared spectroscopy (FT-IR) data of chiral CNPs is illustrated in Figure 2D. The peaks at about 1360~1400 cm -1 and 1600 cm -1 are related to the stretching vibrations of COO and C=C, respectively. The strong absorption band between 3200 cm -1 and 3600 cm -1 can be attributed to the stretching vibrations of -OH and -NH. The FT-IR results indicate that hydroxyl groups, carboxyl groups and amine groups are present on the surface of the chiral CNPs.

[0117] High-concentration sample spots of each CNP enantiomer were spread near the bottom of a reverse-phase (RP) chiral TLC plate made of L-CST bowtie / alumina. A 250 mL beaker with a lid was used as the developing chamber. The TLC plate with the spotted samples was placed vertically in the developing chamber with a small amount of water or methanol as the eluent to saturate with air and equilibrate with the stationary phase. After allowing the mobile phase / sample to move upwards on the column for approximately 5 minutes, the TLC was removed from the chamber to dry and visualized under 254 nm ultraviolet light. An example of a chiral TLC plate and CNP separation can be seen in Figures 3A and 3B. Figure 3A shows a TLC plate with bowtie particles as chiral particles in the active layer of the stationary phase. Figure 3B shows the separation of chiral CNPs on such a stationary phase. The spots on the left are L-CNPs, and the spots on the right are D-CNPs. L-CNPs were observed to have migrated upwards on the TLC plate, while D-CNPs remained as spots.

[0118] In other modifications, stationary phases prepared according to some aspects of this disclosure may be used in column chromatography applications. To prepare an HPLC column or preparative column, the CSP is wet-packed within the column by injection. Pressure may be applied within the column to remove air bubbles and ensure proper packing without degrading the CSP. Beads or other packing media are not required unless the back pressure during HPLC operation is too high. In this case, micrometer-sized beads are suitable as packing media. As described above, these beads or packing media may be silica, polymers, ion-exchange beads, or beads coated with other chiral molecules / nanoparticles.

[0119] In this example, after packing the column with wet-packed CSP, a 5-column length of solvent is passed over the fixed bed to remove air bubbles and ensure uniform packing of the CSP. To ensure uniform packing, a gentle pressure from a pump is used to pack the column. If the column is a preparative column, the sample is loaded into the top (inlet) of the column, and then a gentle pressure is used to drive the separation and collect the fraction at the top. If the column is for HPLC, the column is loaded into the LC instrument, and a pump is used to pass at least a 5-column length of solvent through the system, after which the desired sample is injected into the LC instrument. An example of an HPLC column with alumina / bowtie CSP is shown in Figure 4.

[0120] The above-described embodiments are provided for illustrative and explanatory purposes only and are not intended to be exhaustive or limiting of the disclosure. Individual elements or features of a particular embodiment are generally not limiting to that particular embodiment, but are interchangeable and may be used in selected embodiments where applicable, even if not specifically illustrated or described. Similar elements may also be modified in many ways. Such modifications should not be considered deviations from the disclosure, and all such modifications are intended to be within the scope of the disclosure.

Claims

1. A stationary phase for a chiral chromatography apparatus, which is a stationary phase for separating and / or analyzing one or more target analytes, wherein the stationary phase comprises a porous active layer containing a plurality of nanofibers and a plurality of chiral particles distributed within a matrix.

2. The aforementioned plurality of nanofibers are aluminum oxide (Al ２ O ３ ), carbon, titanium dioxide (TiO ２ ), zinc oxide (ZnO), iron oxide (Fe ２ O ３ ), silicon dioxide (SiO ２ ), zirconium silicate (ZrSiO ４ The stationary phase according to claim 1, comprising a material selected from the group consisting of germanium (Ge), silver (Ag), gold (Au), copper (Cu), or any combination thereof.

3. The plurality of nanofibers described above comprise aluminum oxide (Al ２ O ３ ). The stationary phase according to claim 1.

4. The porous active layer is disposed on a support, as described in claim 1, the stationary phase.

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

6. The stationary phase according to claim 1, wherein the plurality of chiral particles are selected from the group consisting of chiral microparticles including an aggregate of nanoribbons defining a bowtie shape, hedgehog-shaped particles, helices, nanorods, star shapes, cubes, prisms, nanotubes, nanosheets, nanowires, and any combination thereof.

7. The stationary phase according to claim 1, wherein the plurality of chiral particles comprises a plurality of chiral microparticles, each comprising an aggregate of nanoribbons defining a bowtie shape that exhibits chirality.

8. Each nanoribbon of the aggregate contains at least one cadmium ion (Cd ２＋ The stationary phase according to claim 7, comprising at least two peptides interconnected by ).

9. Each of the aforementioned nanoribbons contains at least one cadmium ion (Cd ２＋ The stationary phase according to claim 7, comprising at least two homocystine molecules interconnected by ).

10. The stationary phase according to claim 7, wherein each nanoribbon has a length of about 10 nm to about 100 μm, a thickness of about 50 nm to about 10 μm, and a width of about 3 nm to about 20 μm.

11. The stationary phase according to claim 1, wherein the porous active layer defines a plurality of pores having an average diameter of about 10 nm to about 200 nm.

12. The matrix is ​​calcium sulfate (CaSO4). ４ The stationary phase according to claim 1, comprising a binder material containing ).

13. The stationary phase according to claim 1, wherein the plurality of nanofibers and the plurality of chiral particles are substantially uniformly distributed in the porous active layer.

14. The stationary phase according to claim 1, wherein the plurality of nanofibers have an average diameter of about 10 nm to about 100 μm and an average length of about 100 nm to about 100 μm.

15. The stationary phase according to claim 1, wherein the plurality of chiral particles have an average length of about 100 nm to about 100 μm, an average thickness of about 500 nm to about 10 μm, and an average width of about 50 nm to about 30 μm.

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

17. The stationary phase according to claim 1, wherein at least one portion of the plurality of chiral particles exhibits chirality on multiple scales.

18. A chromatography apparatus for separating and / or analyzing the chirality of one or more target analytes in a sample, The chromatography apparatus described above is An inlet configured to receive the aforementioned sample, A fluid channel comprising a stationary phase and configured to receive the sample as a mobile phase, wherein the stationary phase comprises a porous active layer containing a plurality of nanofibers and a plurality of chiral particles distributed within the matrix, The outlet through which the sample passes, Equipped with, A chromatography apparatus in which the inlet, the fluid channel, and the outlet are in fluid communication with each other.

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

20. The chromatography apparatus according to claim 18, wherein the chromatography apparatus is a chromatography column, and the stationary phase is disposed inside the chromatography column.

21. The chromatography apparatus according to claim 20, wherein the chromatography column is a high-performance liquid chromatography (HPLC) column.

22. The aforementioned plurality of nanofibers are aluminum oxide (Al ２ O ３ ), carbon, titanium dioxide (TiO ２ ), zinc oxide (ZnO), iron oxide (Fe ２ O ３ ), silicon dioxide (SiO ２ ), zirconium silicate (ZrSiO ４ The chromatography apparatus according to claim 18, comprising a material selected from the group consisting of germanium (Ge), silver (Ag), gold (Au), copper (Cu), or any combination thereof.

23. The aforementioned plurality of nanofibers are aluminum oxide (Al ２ O ３ The chromatography apparatus according to claim 18, comprising )

24. The chromatography apparatus according to claim 18, wherein the plurality of chiral particles are selected from the group consisting of microparticles including an aggregate of nanoribbons defining a bowtie shape, hedgehog-shaped particles, helices, nanorods, star shapes, cubes, prisms, nanotubes, nanosheets, nanowires, and any combination thereof.

25. The chromatography apparatus according to claim 18, wherein the plurality of chiral particles include a plurality of chiral microparticles, each comprising an aggregate of nanoribbons that define a bowtie shape exhibiting chirality.

26. Each of the aforementioned nanoribbons contains at least one cadmium ion (Cd ２＋ The chromatography apparatus according to claim 25, comprising at least two homocystine molecules interconnected by ).

27. The chromatography apparatus according to claim 18, wherein the porous active layer defines a plurality of pores having an average diameter of about 10 nm to about 200 nm.

28. The matrix is ​​calcium sulfate (CaSO4). ４ A chromatography apparatus according to claim 18, comprising an inorganic binder material including ).

29. The chromatography apparatus according to claim 18, wherein the plurality of nanofibers and the plurality of chiral particles are substantially uniformly distributed within the porous active layer.

30. The chromatography apparatus according to claim 18, wherein at least one portion of the plurality of chiral particles exhibits chirality on multiple scales.

31. A method for separating and / or analyzing the chirality of one or more target analytes in a chiral chromatography apparatus, wherein the method is: A step of introducing a sample containing at least one target analyte into the inlet of a chiral chromatography apparatus equipped with a fluid channel on which a stationary phase is disposed, wherein the stationary phase includes a porous active layer containing a plurality of nanofibers and a plurality of chiral particles distributed within the matrix, The steps include separating at least one target analyte from a sample by passing the sample through the stationary phase in the fluid channel as a mobile phase in order to determine the chirality of at least one of the target analytes, The steps include eluting the sample from the outlet of the chiral chromatography apparatus, Methods that include...

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

33. The method according to claim 31, wherein the at least one target analyte has a molecular weight of about 10,000 daltons or more.

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

35. The method according to claim 31, wherein the sample further comprises a solvent selected from the group consisting of polar protic solvents, polar aprotic solvents, nonpolar solvents, protic ionic liquids, buffers, and any combination thereof.

36. The method according to claim 31, wherein the sample further comprises an organic modifier for increasing the resolution of the separation, the organic modifier being selected from the group consisting of acetic acid, trifluoroacetic acid (TFA), formic acid, trimethylamine (TEA), and any combination thereof.

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

38. The method according to claim 31, wherein the chiral chromatography apparatus is a chromatography column, and the stationary phase is disposed inside the chromatography column.

39. The method according to claim 38, wherein the chromatography column is a high-performance liquid chromatography (HPLC) column.

40. The method according to claim 31, wherein the plurality of chiral particles are selected from the group consisting of microparticles including an aggregate of nanoribbons defining a bowtie shape, hedgehog-shaped particles, helices, nanorods, star-shaped particles, cubes, prisms, nanotubes, nanosheets, nanowires, and any combination thereof.

41. The method according to claim 31, wherein the plurality of chiral particles include a plurality of chiral microparticles, each comprising an aggregate of nanoribbons defining a bowtie shape that exhibits chirality.

42. The method according to claim 31, wherein at least one portion of the plurality of chiral particles exhibits chirality on multiple scales.

43. The aforementioned plurality of nanofibers are aluminum oxide (Al ２ O ３ ), carbon, titanium dioxide (TiO ２ ), zinc oxide (ZnO), iron oxide (Fe ２ O ３ ), silicon dioxide (SiO ２ ), zirconium silicate (ZrSiO ４ The method according to claim 31, comprising a material selected from the group consisting of germanium (Ge), silver (Ag), gold (Au), copper (Cu), or any combination thereof.

44. The aforementioned plurality of nanofibers are aluminum oxide (Al ２ O ３ The method according to claim 31, including ).

45. The method according to claim 31, further comprising qualitatively evaluating the sample after the separation.

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