Patent Publication Number: US-2019170713-A1

Title: Thin layer chromatography plate and sample analysis method using same

Description:
TECHNICAL FIELD 
     The present disclosure relates to a thin layer chromatography plate and a sample analysis method using the same. 
     BACKGROUND 
     Chromatography and electrophoresis, for example, have been known as a method for separating a specific component from a mixture containing multiple components. Thin layer chromatography, which is a kind of chromatography techniques, makes it possible to easily and quickly separate multiple components from each other. 
     As shown in  FIG. 7 , PTL 1 discloses thin layer chromatography plate  500  provided with first separating agent layer  531  and second separating agent layer  532 . Second separating agent layer  532  is adjacent to first separating agent layer  531 . First separating agent layer  531  and second separating agent layer  532  are respectively formed from separating agents having different optical responses from each other. 
     When thin layer chromatography plate  500  is used, multiple components can be separated from each other as described below. Sample  560  is placed on first separating agent layer  531  and developed in direction X. Then, second separating agent layer  532  is dried. Next, the orientation of thin layer chromatography plate  500  is changed, and sample  560  is developed in direction Y orthogonal to direction X. The multiple components are separated from each other in second separating agent layer  532 . 
     CITATION LIST 
     Patent Literature 
     PTL 1: International Publication No. WO 2011/149041 
     SUMMARY 
     A thin layer chromatography plate according to a first aspect of the present disclosure includes a substrate and a separation layer. The separation layer is disposed on the substrate and configured to separate multiple components included in a sample from each other. In addition, the separation layer includes a first layer and a second layer. The first layer has a porous structure and extends in a first direction. The second layer has a porous structure and extends in the first direction. The first layer and the second layer are arrayed in a second direction orthogonal to the first direction. A zeta potential of the first layer is different from a zeta potential of the second layer. 
     A sample analysis method according to a second aspect of the present disclosure includes the following steps. A sample is placed onto each of the first layer and the second layer of the thin layer chromatography plate according to the first aspect. Each of ends of the first layer and the second layer in the first direction is brought into contact with a developing solvent. 
     According to the thin layer chromatography plate in the present disclosure and a sample analysis method using the same, a sample can be analyzed more easily and more quickly. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1A  is a plan view illustrating a thin layer chromatography plate according to a first exemplary embodiment. 
         FIG. 1B  is a sectional view illustrating the thin layer chromatography plate shown in  FIG. 1A  along line IB-IB. 
         FIG. 2A  is a partially enlarged sectional view illustrating the thin layer chromatography plate according to the first exemplary embodiment. 
         FIG. 2B  is a partially enlarged sectional view illustrating a thin layer chromatography plate according to a modification of the first exemplary embodiment. 
         FIG. 2C  is a partially enlarged sectional view illustrating a thin layer chromatography plate according to another modification of the first exemplary embodiment. 
         FIG. 3A  is a schematic view showing a state where a sample is placed on the thin layer chromatography plate according to the first exemplary embodiment. 
         FIG. 3B  is a schematic view showing a state where the thin layer chromatography plate shown in  FIG. 3A  is brought into contact with a developing solvent. 
         FIG. 4A  is a plan view illustrating a thin layer chromatography plate according to a second exemplary embodiment. 
         FIG. 4B  is a sectional view illustrating the thin layer chromatography plate shown in  FIG. 4A  along line IVB-IVB. 
         FIG. 5A  is a plan view illustrating a thin layer chromatography plate according to a third exemplary embodiment. 
         FIG. 5B  is a sectional view illustrating the thin layer chromatography plate shown in  FIG. 5A  along line VB-VB. 
         FIG. 6A  is a plan view illustrating a thin layer chromatography plate according to a fourth exemplary embodiment. 
         FIG. 6B  is a sectional view illustrating the thin layer chromatography plate shown in  FIG. 6A  along line VIB-VIB. 
         FIG. 7  is a plan view illustrating a conventional thin layer chromatography plate. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     According to the method disclosed in PTL 1, it is necessary that, after a sample is developed in first separating agent layer  531 , the orientation of thin layer chromatography plate  500  is changed and the sample is developed in second separating agent layer  532 . The present disclosure provides a technique for analyzing a sample more easily and more quickly. 
     (Underlying Knowledge of the Present Disclosure) 
     A condition of human skin can be checked by analyzing proteins included in the human skin. The protein analysis is conducted in the manner described below, for example. A sample such as an epiderm is extracted from a skin of an examinee. The sample includes multiple kind of proteins. The multiple kind of proteins included in the sample are separated from each other using thin layer chromatography. Each of the separated proteins is identified. 
     For example, if the sample includes a protein caused by rough skin, it is found that the examinee has rough skin. If it is possible to recognize a condition of the skin of the examinee, cosmetics suitable for the examinee can be recommended. It is convenient to check the condition of the skin of the examinee and recommend cosmetics based on the check result in cosmetics retail stores. When doing so, protein analysis needs to be quickly conducted during a waiting time of the examinee. 
     The thin layer chromatography plate according to the present disclosure includes a substrate and a separation layer. The separation layer is disposed on the substrate and configured to separate multiple components included in a sample from each other. In addition, the separation layer includes a first layer and a second layer. The first layer has a porous structure and extends in a first direction. The second layer has a porous structure and extends in the first direction. The first layer and the second layer are arrayed in a second direction orthogonal to the first direction. A zeta potential of the first layer is different from a zeta potential of the second layer. 
     According to the present disclosure, the zeta potential of the first layer is different from the zeta potential of the second layer, whereby an interaction between the multiple components included in the sample and the first layer is different from an interaction between the multiple components and the second layer. Therefore, when the multiple components are developed in the first layer and the second layer, different results can be obtained in the first layer and the second layer. For example, the multiple components which are not separated from each other in the first layer are separated from each other in the second layer. The multiple components which are not separated from each other in the second layer are separated from each other in the first layer. Therefore, each of the multiple components can be identified based on the development result of the multiple components in a first stage, which eliminates a need to develop the multiple components in a second stage. Thus, the sample can be analyzed more easily and more quickly. 
     The thin layer chromatography plate described above may be configured such that the first layer includes a first metal oxide, the second layer includes a second metal oxide, and the first metal oxide has an isoelectric point different from an isoelectric point of the second metal oxide, for example. With this configuration, the zeta potential of the first layer can be different from the zeta potential of the second layer. Further, only the second layer among the first layer and the second layer may has the porous structure modified with a metal oxide. With this configuration, the zeta potential of the first layer can be different from the zeta potential of the second layer. 
     In addition, the first metal oxide in the thin layer chromatography plate is disposed on the porous structure of the first layer, for example. With this configuration, the interaction between the multiple components included in the sample and the first layer is accelerated. Thus, the multiple components may be easily separated from each other in the first layer. 
     In addition, the first layer of the thin layer chromatography plate includes a first metal oxide film disposed on the porous structure of the first layer. And the first metal oxide film is made of the first metal oxide, for example. With this configuration, the interaction between the multiple components included in the sample and the first layer is accelerated. Thus, the multiple components may be easily separated from each other in the first layer. 
     In addition, the porous structure of the first layer of the thin layer chromatography plate includes an aggregate of particles coated with a first metal oxide film. And the first metal oxide film is made of the first metal oxide, for example. With this configuration, the interaction between the multiple components included in the sample and the first layer is accelerated. Thus, the multiple components may be easily separated from each other in the first layer. 
     Further, the second metal oxide of the thin layer chromatography plate is disposed on the porous structure of the second layer, for example. With this configuration, the interaction between the multiple components included in the sample and the second layer is accelerated. Thus, the multiple components may be easily separated from each other in the second layer. 
     In addition, the second layer of the thin layer chromatography plate includes a second metal oxide film disposed on the porous structure of the second layer. And the second metal oxide film is made of the second metal oxide, for example. With this configuration, the interaction between the multiple components included in the sample and the second layer is accelerated. Thus, the multiple components may be easily separated from each other in the second layer. 
     In addition, the porous structure of the second layer of the thin layer chromatography plate includes an aggregate of particles coated with a second metal oxide film. And the second metal oxide film is made of the second metal oxide, for example. With this configuration, the interaction between the multiple components included in the sample and the second layer is accelerated. Thus, the multiple components may be easily separated from each other in the second layer. 
     In addition, the porous structure of the first layer of the thin layer chromatography plate includes an aggregate of particles each of which has a single composition phase, for example. 
     Further, the first metal oxide in the thin layer chromatography plate includes at least one selected from the group consisting of titanium oxide, aluminum oxide, tin oxide, zinc oxide, tungsten oxide, manganese oxide, nickel oxide, copper oxide, and magnesium oxide, for example. Thus, the multiple components may be easily separated from each other in the first layer. 
     Further, the second metal oxide in the thin layer chromatography plate includes at least one selected from the group consisting of titanium oxide, aluminum oxide, tin oxide, zinc oxide, tungsten oxide, manganese oxide, nickel oxide, copper oxide, and magnesium oxide, for example. Thus, the multiple components may be easily separated from each other in the second layer. 
     In addition, the first layer and the second layer of the thin layer chromatography plate are in contact with each other, for example. With this configuration, the separation layer can be easily manufactured. 
     In addition, the thin layer chromatography plate further includes a functional layer having a band shape. The functional layer is disposed on the separation layer and on which the sample is to be placed. The functional layer extends in the second direction, for example. Accordingly, when the sample is placed on the functional layer, the sample penetrates into the functional layer. The sample spreads all over the functional layer. The sample penetrating into the functional layer is brought into contact with the separation layer. Thus, it is unnecessary to place the sample on the separation layer several times. Accordingly, the sample can be efficiently placed on the separation layer. 
     The sample analysis method according to the present disclosure includes placing a sample on each of a first layer and a second layer of the thin layer chromatography plate according to the present disclosure, and bringing each of ends of the first layer and the second layer in a first direction into contact with a developing solvent. 
     Thus, different results can be obtained in the first layer and the second layer. For example, the multiple components which are not separated from each other in the first layer are separated from each other in the second layer. The multiple components which are not separated from each other in the second layer are separated from each other in the first layer. Therefore, each of the multiple components can be identified based on the development result of the multiple components in a first stage, which eliminates a need to develop the multiple components in a second stage. Thus, the sample can be analyzed more easily and more quickly. 
     Further, the developing solvent used in the sample analysis method contains water, for example. With this configuration, when the first layer and the second layer are brought into contact with the developing solvent, the first metal oxide and the second metal oxide are charged. Types of charges or amounts of charges generated in the first metal oxide and the second metal oxide are different from each other. Therefore, the interaction between the multiple components included in the sample and the first layer is greatly different from the interaction between the multiple components and the second layer. Thus, each of the multiple components can be easily identified based on the development result of the multiple components in the first stage. 
     In addition, the sample used in the sample analysis method includes a protein, for example. With this configuration, the interaction between the first layer or the second layer and the protein may be accelerated. Thus, the multiple components can be easily separated from each other in the first layer or the second layer. 
     Exemplary embodiments of the present disclosure will be described below with reference to the drawings. The present disclosure is not limited to the following exemplary embodiments. 
     First Exemplary Embodiment 
     As shown in  FIGS. 1A and 1B , thin layer chromatography plate  100  (hereinafter referred to as “TLC plate  100 ”) according to the present exemplary embodiment has substrate  10  and separation layer  20 . Substrate  10  has a plate shape, for example. Substrate  10  has, for example, a rectangular shape in a plan view. Separation layer  20  is disposed on substrate  10 . Separation layer  20  covers the surface of substrate  10 . Substrate  10  has two pairs of end faces facing each other. In the present exemplary embodiment, development direction X (first direction) extends from one end face of one pair of the two pairs of end faces of substrate  10  to the other end face. Multiple components included in the sample are developed in development direction X. Array direction Y (second direction) extends from one end face of another pair of the two pairs of end faces of substrate  10  to the other end face and is orthogonal to development direction X. 
     Separation layer  20  separates the multiple components included in the sample from each other. Separation layer  20  includes first layer  31  and second layer  32 . First layer  31  is a layer having a band shape. First layer  31  has a rectangular band shape in a plan view. First layer  31  extends in development direction X. First layer  31  extends from one of a pair of end faces of substrate  10  to the other in development direction X. Note that first layer  31  may not reach to the other end face of substrate  10 . 
     Second layer  32  is a layer having a band shape. Second layer  32  has a rectangular band shape in a plan view. Second layer  32  extends in development direction X. Second layer  32  extends from one of a pair of end faces of substrate  10  to the other in development direction X. Note that second layer  32  may not reach to the other end face of substrate  10 . 
     First layer  31  and second layer  32  are both disposed on substrate  10 . In other words, first layer  31  and second layer  32  are both in contact with substrate  10 . First layer  31  and second layer  32  are arrayed in array direction Y. In the present exemplary embodiment, second layer  32  is in contact with first layer  31 . A side surface of first layer  31  and a side surface of second layer  32  are in contact with each other. When separation layer  20  is viewed in a plan view, one side of first layer  31  is in contact with one side of second layer  32 . A length of the one side of first layer  31  is equal to a length of the one side of second layer  32 . Boundary face  40  is formed due to the contact between first layer  31  and second layer  32 . Boundary face  40  extends in development direction X. Note that second layer  32  may not be in contact with first layer  31 . 
     The material of substrate  10  is not particularly limited, as long as it can maintain the shape of TLC plate  100  without being eluted in a developing solvent. The material of substrate  10  is glass, resin, metal, or paper, for example. Substrate  10  is typically a glass plate or an aluminum film. 
     First layer  31  has a porous structure. The porous structure of first layer  31  can carry the developing solvent from one end to the other end of first layer  31  in development direction X due to capillary force. The material of the porous structure is not particularly limited. The material of the porous structure includes at least one selected from the group consisting of a fiber material, an inorganic material, and a polymer material, for example. 
     The fiber material includes at least one selected from the group consisting of a plant fiber, an animal fiber, a recycled fiber, a synthetic fiber, and a glass fiber, for example. The plant fiber includes cellulose, for example. The synthetic fiber includes cellulose acetate, for example. 
     The inorganic material includes at least one selected from the group consisting of alumina, silicon dioxide, and zirconia, for example. The polymer material includes at least one selected from the group consisting of agarose, dextran, mannan, fluororesin, polystyrene, polyethylene, polypropylene, polyurethane, and polyvinyl chloride, for example. 
     The porous structure is formed from at least one selected from the group consisting of filter paper, an aggregate of inorganic particles, a porous body of a polymer material, and an aggregate of polymer material particles, for example. The inorganic particles include at least one kind selected from the group consisting of alumina particles, silica gel particles, silicon pillar, zeolite particles, diatomaceous earth, and zirconia particles, for example. The inorganic particles may be modified with a hydrophobic functional group or a hydrophilic functional group. The hydrophobic functional group includes a functional group having a hydrocarbon group at the end, for example. The hydrocarbon group includes at least one selected from the group consisting of an octadecyl group, an octyl group, a t-butyl group, a trimethylsilyl group, and a phenyl group, for example. The hydrophilic functional group includes at least one selected from the group consisting of a functional group having a cyano group and a functional group having an amino group, for example. 
     An average pore diameter of the porous structure of first layer  31  may range from 0.01 μm to 100 μm both inclusive. When the porous structure of first layer  31  is formed from an aggregate of inorganic particles or polymer material particles, an average particle diameter of inorganic particles or polymer material particles may range from 1 μm to 100 μm both inclusive. The “average pore diameter” can be measured with the following method. Specifically, the surface or cross-section of first layer  31  is observed with an electron microscope (for example, an electron scanning microscope). Pore diameters of a plurality of pores (for example, random  50  pores) in the observed porous structure are measured. The average pore diameter is determined based on the average value calculated using the measured values. The diameter of a circle having an area equal to the area of the pore observed with the electron microscope can be regarded as the pore diameter. The “average particle diameter” can be measured with the following method. Specifically, the surface or cross-section of first layer  31  is observed with an electron microscope, and diameters of random number (for example, 50) of particles constituting the porous structure of first layer  31  are measured. The average particle diameter is determined based on the average value calculated using the obtained measured values. The diameter of a circle having an area equal to the area of the particle observed with the electron microscope can be regarded as the particle diameter. 
     First layer  31  includes a first metal oxide. The first metal oxide is not particularly limited, as long as it is a metal oxide. The first metal oxide includes at least one selected from the group consisting of titanium oxide, aluminum oxide, tin oxide, zinc oxide, tungsten oxide, manganese oxide, nickel oxide, copper oxide, and magnesium oxide, for example. The first metal oxide may be a semimetal oxide. The semimetal oxide includes at least one selected from the group consisting of boron oxide and silicon dioxide, for example. The first metal oxide is different from the material of the porous structure of first layer  31 . The composition of the first metal oxide is different from the composition of the porous structure of first layer  31 . The first metal oxide may be included in the porous structure of first layer  31 . 
     The first metal oxide may be in contact with a part of the porous structure of first layer  31 . The first metal oxide may be disposed on the porous structure of first layer  31 . The first metal oxide may be disposed between the porous structure of first layer  31  and substrate  10 . When the porous structure of first layer  31  is formed from an aggregate of inorganic particles or polymer material particles, the first metal oxide may be located between multiple particles constituting the porous structure of first layer  31 . As shown in  FIG. 2A , first metal oxide film  81  may be disposed on porous structure  80  of first layer  31 . First metal oxide film  81  is made of the first metal oxide. As shown in  FIG. 2B , first metal oxide film  81  may be disposed between porous structure  80  of first layer  31  and substrate  10 . When the first metal oxide is in contact with porous structure  80  of first layer  31 , the interaction between the multiple components included in the sample and first layer  31  is accelerated. Thus, the multiple components may be easily separated from each other in first layer  31 . In  FIG. 2A , first metal oxide film  81  may partially cover the surface of porous structure  80 . In  FIG. 2B , first metal oxide film  81  may partially cover the surface of substrate  10 . 
     As shown in  FIG. 2C , porous structure  80  of first layer  31  may include an aggregate of particles coated with first metal oxide film  81 . In  FIG. 2C , first layer  31  is constituted by an aggregate of particles each of which is coated with first metal oxide film  81 . The particles include at least one kind selected from the group consisting of inorganic particles and polymer material particles, for example. First metal oxide film  81  may coat the entire surface of the particle or coat a part of the surface of the particle. When porous structure  80  includes an aggregate of particles coated with first metal oxide film  81 , the interaction between the multiple components included in the sample and first layer  31  is accelerated. Thus, the multiple components may be easily separated from each other in first layer  31 . 
     Whether each of the particles is coated with first metal oxide film  81  can be confirmed by observing the cross-section of first layer  31  with an electron microscope (for example, an electron scanning microscope). Whether each of the particles is coated with first metal oxide film  81  can also be confirmed by conducting an elemental analysis on the cross-section of first layer  31 . The elemental analysis can be conducted by X-ray photoelectron spectroscopy (XPS) or energy dispersive X-ray spectroscopy (EDX), for example. 
     The thickness of first metal oxide film  81  is not particularly limited. The thickness of first metal oxide film  81  is determined according to the material of first metal oxide film  81 , for example. There is a tendency that, as first metal oxide film  81  is thicker, the multiple components are more easily separated from each other in first layer  31  when the sample is developed. As first metal oxide film  81  is thicker, the mobility of the developing solvent in first layer  31  slows down. The thickness of first metal oxide film  81  ranges from 10 nm to 1000 nm both inclusive, for example. 
     First layer  31  may further include an additive. Examples of the additive include a fluorescence indicator and a binder. 
     Examples of the fluorescence indicator include magnesium tungstate and zinc silicate containing manganese. When first layer  31  includes the fluorescence indicator, positions of the multiple components can be detected by irradiating first layer  31  with ultraviolet ray. 
     The binder includes at least one selected from the group consisting of an inorganic binder, an organic fiber, a thickener, and an organic binder, for example. Examples of the inorganic binder include plaster and colloidal silica. Examples of the organic fiber include microfibrillar cellulose. Examples of the thickener include hydroxyethyl cellulose and carboxymethyl cellulose. Examples of the organic binder include polyvinyl alcohol and polyacrylic acid. When first layer  31  includes the binder, adhesiveness between substrate  10  and first layer  31  is improved. When the porous structure of first layer  31  is formed from an aggregate of inorganic particles or polymer material particles, durability of the aggregate of inorganic particles or polymer material particles is improved due to the binder. 
     The above-mentioned additives may be mixed into the material of the porous structure of first layer  31 . The additives may coat the surfaces of inorganic particles or polymer material particles constituting the porous structure. 
     Second layer  32  has a porous structure. The porous structure of second layer  32  can carry the developing solvent from one end to the other end of second layer  32  in development direction X due to capillary force. The material of the porous structure of second layer  32  may be the same as any of those described as examples of the material of the porous structure of first layer  31 . An average pore diameter of the porous structure of second layer  32  may range from 0.01 μm to 100 μm both inclusive. When the porous structure of second layer  32  is formed from an aggregate of inorganic particles or polymer material particles, an average particle diameter of inorganic particles or polymer material particles may range from 1 μm to 100 μm both inclusive. 
     Second layer  32  includes a second metal oxide. The second metal oxide is not particularly limited, as long as it is a metal oxide. The second metal oxide includes at least one selected from the group consisting of titanium oxide, aluminum oxide, tin oxide, zinc oxide, tungsten oxide, manganese oxide, nickel oxide, copper oxide, and magnesium oxide, for example. The second metal oxide may be a semimetal oxide. The semimetal oxide includes at least one selected from the group consisting of boron oxide and silicon dioxide, for example. The second metal oxide is different from the material of the porous structure of second layer  32 . The composition of the second metal oxide is different from the composition of the porous structure of second layer  32 . The second metal oxide may be included in the porous structure of second layer  32 . 
     The second metal oxide is different from the first metal oxide. Specifically, an isoelectric point of the second metal oxide is different from an isoelectric point of the first metal oxide. A difference between the isoelectric point of the first metal oxide and the isoelectric point of the second metal oxide is 1 to 8, for example. The “isoelectric point” can be measured with the following method. Specifically, a water containing solvent and a metal oxide are brought into contact with each other. A zeta potential on the surface of the metal oxide is measured. The zeta potential can be measured by a commercially available zeta potential measurement device, for example. The pH value of the solvent when the zeta potential on the surface of the metal oxide becomes zero can be regarded as the isoelectric point of the metal oxide. The isoelectric point of the metal oxide is determined by the metal oxide. For example, the isoelectric point of tin oxide is typically 4.5 to 7.3. The isoelectric point of zinc oxide is typically 9.2. The isoelectric point of tungsten oxide is typically 0.5. The isoelectric point of nickel oxide is typically 10.3±0.4. The isoelectric point of magnesium oxide is typically 12.4±0.3. The isoelectric point of silicon dioxide is typically 1.8 to 2.2. 
     The second metal oxide may be in contact with a part of the porous structure of second layer  32 . The second metal oxide may be disposed on the porous structure of second layer  32 . The second metal oxide may be disposed between the porous structure of second layer  32  and substrate  10 . When the porous structure of second layer  32  is formed from an aggregate of inorganic particles or polymer material particles, the second metal oxide may be located between multiple particles constituting the porous structure of second layer  32 . A second metal oxide film may be disposed on the porous structure of second layer  32 . The second metal oxide film is made of the second metal oxide. The second metal oxide film may partially cover the surface of the porous structure of second layer  32 . The second metal oxide film may be disposed between the porous structure of second layer  32  and substrate  10 . The second metal oxide film may partially cover the surface of substrate  10 . When the second metal oxide is in contact with the porous structure of second layer  32 , the interaction between the multiple components included in the sample and second layer  32  is accelerated. Thus, the multiple components may be easily separated from each other in second layer  32 . 
     The porous structure of second layer  32  may include an aggregate of particles coated with the second metal oxide film. Second layer  32  may be formed from an aggregate of particles coated with the second metal oxide film. The particles include at least one kind selected from the group consisting of inorganic particles and polymer material particles, for example. The second metal oxide film may coat the entire surface of the particle or coat a part of the surface of the particle. When the porous structure includes an aggregate of particles coated with the second metal oxide film, the interaction between the multiple components included in the sample and second layer  32  is accelerated. Thus, the multiple components may be easily separated from each other in second layer  32 . 
     The thickness of the second metal oxide film is not particularly limited. The thickness of the second metal oxide film is determined according to the material of the second metal oxide film, for example. There is a tendency that, as the second metal oxide film is thicker, the multiple components are more easily separated from each other in second layer  32  when the sample is developed. As the second metal oxide film is thicker, the mobility of the developing solvent in second layer  32  slows down. The thickness of the second metal oxide film ranges from 10 nm to 1000 nm both inclusive, for example. 
     Second layer  32  may further include any of the above-mentioned additives. 
     Length L 1  of first layer  31  in development direction X is not particularly limited. Length L 1  is determined according to the material of the porous structure of first layer  31 , the first metal oxide, and a size of a container for housing TLC plate  100 , for example. Length L 1  ranges from 20 mm to 200 mm both inclusive, for example. The length of second layer  32  and the length of substrate  10  in development direction X are typically equal to length L 1 . 
     Length L 2  of first layer  31  in array direction Y is not particularly limited. Length L 2  is determined according to an amount of the sample to be placed on first layer  31 , for example. Length L 2  ranges from 10 mm to 100 mm both inclusive, for example. 
     Length L 3  of second layer  32  in array direction Y is not particularly limited. Length L 3  is equal to a value of length L 2 . The length of substrate  10  in development direction X is equal to the total of length L 2  and length L 3 . 
     Thickness L 4  of first layer  31  is not particularly limited. Thickness L 4  is determined according to the porous structure of first layer  31 , and the first metal oxide, for example. Thickness L 4  ranges from 0.05 mm to 1 mm both inclusive, for example. The thickness of second layer  32  is typically equal to thickness L 4  of first layer  31 . 
     Thickness L 5  of substrate  10  is not particularly limited as long as the shape of TLC plate  100  can be maintained. Thickness L 5  ranges from 0.1 mm to 5 mm both inclusive, for example. 
     Next, a manufacturing method of TLC plate  100  will be described. 
     First, a first dispersion liquid containing inorganic particles or polymer material particles is prepared. The first dispersion liquid can be obtained by dispersing inorganic particles or polymer material particles into a coating solvent. 
     The coating solvent includes at least one selected from the group consisting of water and an organic solvent, for example. The organic solvent includes at least one selected from the group consisting of alcohol, ketone, ether, nitrile, sulfoxide, sulfone, ester, carboxylic acid, amide, hydrocarbon, aromatic hydrocarbon, and halogen-containing compound, for example. Examples of alcohol include methanol, ethanol, and isopropyl alcohol. Examples of ketone include acetone and ethyl methyl ketone. Examples of ether include tetrahydrofuran and dioxane. Examples of nitrile include acetonitrile. Examples of sulfoxide include dimethyl sulfoxide. Examples of sulfone include sulfolane. Examples of ester include ethyl acetate. Examples of carboxylic acid includes formic acid and acetic acid. Examples of amide include dimethylformamide. Examples of hydrocarbon include pentane and hexane. Examples of aromatic hydrocarbon include benzene, toluene, and xylene. Examples of halogen-containing compound include methylene chloride, chloroform, bromoform, chlorobenzene, and bromobenzene. 
     The first dispersion liquid is applied on a part of the surface of substrate  10  to form a coating film. The coating film is dried, whereby an untreated layer of first layer  31  is formed on substrate  10 . The untreated layer of first layer  31  may be formed on substrate  10  by bonding filter paper or a porous body of polymer materials on a part of the surface of substrate  10  under pressure. 
     Then, a first metal oxide is deposited on the untreated layer of first layer  31 . Thus, first layer  31  is formed on substrate  10 . In this case, a first metal oxide film may be formed by the deposition of the first metal oxide on the untreated layer. Examples of a method for depositing the first metal oxide include sputtering using an existing mask, ion plating, electron beam evaporation, vacuum deposition, chemical vapor deposition, and chemical vapor deposition. In the manufacturing method in the present exemplary embodiment, the first metal oxide is deposited after the untreated layer of first layer  31  is formed, whereby first layer  31  is easily formed. 
     The first metal oxide may be deposited on substrate  10  in advance. In this case, the first metal oxide film may be formed by the deposition of the first metal oxide on substrate  10 . The first dispersion liquid is applied on the deposited first metal oxide, and the obtained coating film is dried. Thus, first layer  31  is formed on substrate  10 . First layer  31  may be formed on substrate  10  by bonding filter paper or a porous body of polymer materials on the first metal oxide under pressure. 
     The first dispersion liquid may include the first metal oxide. The first dispersion liquid may include particles coated with the first metal oxide film. Particles coated with the first metal oxide film can be manufactured by the following method, for example. A metal salt is dissolved in the first dispersion liquid. The metal salt is at least a salt of one metal selected from the group consisting of titanium, aluminum, tin, zinc, tungsten, manganese, nickel, copper, and magnesium, for example. When the metal salt is dissolved in the first dispersion liquid, a complex compound is generated. The complex compound adheres to the surfaces of the inorganic particles or polymer material particles. The inorganic particles or polymer material particles to which the complex compound adheres are treated such that metal oxides are deposited. The treatment includes, for example, changing pH of the first dispersion liquid or oxidation of the complex compound. The oxidation of the complex compound is conducted by heating the inorganic particles or polymer material particles, for example. Thus, particles coated with the first metal oxide film are obtained. 
     Then, a second dispersion liquid containing the inorganic particles or polymer material particles is prepared. The second dispersion liquid can be obtained by dispersing inorganic particles or polymer material particles into a coating solvent. The materials mentioned above can be used for the coating solvent. 
     The second dispersion liquid is applied on a part of the surface of substrate  10  to form a coating film. The coating film is dried, whereby an untreated layer of second layer  32  is formed on substrate  10 . The untreated layer of second layer  32  may be formed on substrate  10  by bonding filter paper or a porous body of polymer materials on a part of the surface of substrate  10  under pressure. 
     Then, a second metal oxide is deposited on the untreated layer of second layer  32 . Thus, second layer  32  is formed on substrate  10 . In this case, a second metal oxide film may be formed by the deposition of the second metal oxide on the untreated layer. The methods mentioned above can be used for depositing the second metal oxide. In the manufacturing method in the present exemplary embodiment, the second metal oxide is deposited after the untreated layer of second layer  32  is formed, whereby second layer  32  is easily formed. 
     The second metal oxide may be deposited on substrate  10  in advance. In this case, the second metal oxide film may be formed by the deposition of the second metal oxide on substrate  10 . The second dispersion liquid is applied on the deposited second metal oxide, and the obtained coating film is dried. Thus, second layer  32  is formed on substrate  10 . Second layer  32  may be formed on substrate  10  by bonding filter paper or a porous body of polymer materials on the second metal oxide under pressure. 
     The second dispersion liquid may include the second metal oxide. The second dispersion liquid may include particles coated with the second metal oxide film. The above-mentioned methods for manufacturing particles coated with the first metal oxide film can be used as a method for manufacturing particles coated with the second metal oxide film, for example. 
     First layer  31  and second layer  32  may be formed by the following method. The first dispersion liquid is applied on the entire surface of substrate  10  to form a coating film. The coating film is dried, whereby an untreated layer of first layer  31  and an untreated layer of second layer  32  are formed on substrate  10 . A first metal oxide is deposited on the untreated layer of first layer  31 . A second metal oxide is deposited on the untreated layer of second layer  32 . Thus, first layer  31  and second layer  32  are formed on substrate  10 . The first metal oxide and the second metal oxide are deposited after the untreated layer of first layer  31  and the untreated layer of second layer  32  are formed, whereby separation layer  20  can be easily formed. In separation layer  20  formed by the above method, the side surface of first layer  31  is in contact with the side surface of second layer  32 . 
     The order of formation of first layer  31  and second layer  32  on substrate  10  is not particularly limited. First layer  31  may be formed on substrate  10  after second layer  32  is formed on substrate  10 . 
     Next, the sample analysis method using TLC plate  100  will be described. 
     First, sample  60  is placed on each of first layer  31  and second layer  32  of separation layer  20  of TLC plate  100 , as shown in  FIG. 3A . When sample  60  is placed on first layer  31 , sample  60  penetrates into first layer  31 , so that circular spot  61  is formed. When sample  60  is placed on second layer  32 , sample  60  penetrates into second layer  32 , so that circular spot  62  is formed. Sample  60  is an aqueous solution containing a plurality of proteins, for example. The proportion of the plurality of proteins in sample  60  ranges from 0.01 wt. % to 1 wt. % both inclusive, for example. The volume of sample  60  placed on each of first layer  31  and second layer  32  ranges from 0.5 μL to 10 μL both inclusive, for example. The position where sample  60  is to be placed on each of first layer  31  and second layer  32  is not particularly limited, as long as sample  60  is not in direct contact with the developing solvent. An end of first layer  31  in development direction X is defined as end  31   a , and an end of second layer  32  in development direction X is defined as end  32   a . The distance from end  31   a  to the gravity center of spot  61  in development direction X may be equal to the distance from end  32   a  to the gravity center of spot  62  in development direction X. 
     Then, as shown in  FIG. 3B , TLC plate  100  is placed in container  75  with end  31   a  of first layer  31  and end  32   a  of second layer  32  being directed downward. Container  75  contains developing solvent  70 . Container  75  is a glass jar, for example. Container  75  may be installed inside an analyzing device (not shown). 
     Developing solvent  70  is not particularly limited, as long as it can proceed in first layer  31  or second layer  32  due to capillary force when being brought into contact with first layer  31  or second layer  32 . Developing solvent  70  may contain water. When containing water, developing solvent  70  may contain water in a proportion ranging from 20 wt. % to 100 wt. % both inclusive. When developing solvent  70  contains water and sample  60  contains proteins, solubility of the proteins in developing solvent  70  is improved. Developing solvent  70  may contain an organic solvent. The materials mentioned above as examples of the coating solvent can be used as the organic solvent. The organic solvent contains at least one selected from the group consisting of methanol, ethanol, isopropyl alcohol, acetonitrile, and acetic acid, for example. When containing an organic solvent, developing solvent  70  may contain the organic solvent in a proportion ranging from 20 wt. % to 100 wt. % both inclusive. When developing solvent  70  contains carboxylic acid and sample  60  contains proteins, the frequency of absorption and desorption of proteins to and from each of the porous structure of first layer  31  and the porous structure of second layer  32  is improved. Developing solvent  70  may be an aqueous solution. A solute of the aqueous solution contains at least one selected from the group consisting of phosphate, citrate, acetate, and borate, for example. 
     When TLC plate  100  is placed in container  75 , end  31   a  of first layer  31  and end  32   a  of second layer  32  are in contact with developing solvent  70 . The liquid level of developing solvent  70  is set to prevent direct contact between developing solvent  70  and sample  60 . Due to the capillary force, developing solvent  70  proceeds in development direction X from end  31   a  of first layer  31  and end  32   a  of second layer  32 . When developing solvent  70  is brought into contact with sample  60 , the multiple components included in sample  60  are dissolved into developing solvent  70 . The multiple components dissolved in developing solvent  70  move in development direction X along with developing solvent  70 . The multiple components located in spot  61  move while repeatedly adsorbed and desorbed to and from the porous structure of first layer  31 . Since the frequency of adsorption and desorption varies according to each component, the multiple components are separated from each other in first layer  31 . The multiple components located in spot  62  move while repeatedly adsorbed and desorbed to and from the porous structure of second layer  32 . Since the frequency of adsorption and desorption varies according to each component, the multiple components are separated from each other in second layer  32 . 
     A method for detecting positions of multiple components is not particularly limited, and any known methods can be employed. For example, when first layer  31  and second layer  32  contain a fluorescence indicator, separation layer  20  may be irradiated with ultraviolet ray to detect the positions of multiple components. In such a case, each of the multiple components can be a compound that absorbs ultraviolet ray. The analyzing device may have a mechanism for emitting ultraviolet ray. The positions of the multiple components may be detected by depositing a coloring reagent onto separation layer  20 . In such a case, TLC plate  100  may be heated as necessary. Any known coloring reagent can be used. Examples of the coloring reagent include anisaldehyde, phosphomolybdic acid, iodine, ninhydrin, chameleon solution, 2,4dinitrophenylhydrazine, manganese chloride, and bromocresol green. 
     Under the same condition, the positions of the multiple components after sample  60  is developed are determined for each component. Therefore, with the sample analysis method according to the present exemplary embodiment, each of the separated multiple components can be identified. For example, a component having a known structure is developed on TLC plate  100  under the condition same as the condition for developing sample  60 . Data in which the position of the component after the development and the structure of the component are associated with each other is acquired. This data may be stored in a memory of the analyzing device in advance. Through comparison with the data, each of the multiple components can be identified based on the position of each component after sample  60  is developed. 
     In TLC plate  100 , the isoelectric point of the first metal oxide included in first layer  31  is different from the isoelectric point of the second metal oxide included in second layer  32 . Specifically, the interaction between the multiple components included in sample  60  and first layer  31  is different from the interaction between the multiple components and second layer  32 . Therefore, when the multiple components are developed in first layer  31  and second layer  32 , different results can be obtained in first layer  31  and second layer  32 . For example, the multiple components which are not separated from each other in first layer  31  are separated from each other in second layer  32 . The multiple components which are not separated from each other in second layer  32  are separated from each other in first layer  31 . Each of the multiple components can be identified based on the development result of the multiple components in a first stage. Therefore, it is unnecessary to develop the multiple components in a second stage. Thus, sample  60  can be analyzed more easily and more quickly. 
     When developing solvent  70  contains water, the first metal oxide and the second metal oxide are charged due to contact with developing solvent  70 . Specifically, when the pH of developing solvent  70  is smaller than the isoelectric point of the metal oxide, the metal oxide is positively charged. When the pH of developing solvent  70  is greater than the isoelectric point of the metal oxide, the metal oxide is negatively charged. The isoelectric point of the first metal oxide and the isoelectric point of the second metal oxide are different from each other, and thus, types or amounts of charges generated in the first metal oxide and the second metal oxide differ from each other. Accordingly, the interaction between the multiple components and first layer  31  greatly differs from the interaction between the multiple components and second layer  32 . Thus, each of the multiple components can be identified based on the development result of the multiple components in the first stage. 
     When sample  60  contains a protein, the interaction between first layer  31  or second layer  32  and the protein may be accelerated. Specifically, a specific functional group contained in the protein may be coordinated to the first metal oxide or the second metal oxide. For example, when the protein has a phosphate group, the phosphate group is coordinated to titanium oxide. When the protein has a sugar chain, the sugar chain is coordinated to boron oxide. Therefore, when a metal oxide to which a specific functional group included in the protein can be coordinated is selected as the first metal oxide or the second metal oxide, the multiple components can be easily separated from each other in first layer  31  or second layer  32 . 
     Depending on the multiple components included in sample  60 , first layer  31  may not include the first metal oxide. Similarly, second layer  32  may not include the second metal oxide. In such a case, TLC plate  100  needs to satisfy at least one requirement selected from among the requirement in which the composition of first layer  31  is different from the composition of second layer  32  and the requirement in which the structure of first layer  31  is different from the structure of second layer  32 . When the above requirement is satisfied, the interaction between the multiple components included in sample  60  and first layer  31  is different from the interaction between the multiple components and second layer  32 . Therefore, when the multiple components are developed in first layer  31  and second layer  32 , different results can be obtained in first layer  31  and second layer  32 . “The structure of first layer  31  being different from the structure of second layer  32 ” means that at least one selected from among an average pore diameter of the porous structure of first layer  31 , a void ratio of the porous structure, and an average particle diameter of the material of the porous structure is different from that of the porous structure of second layer  32 , for example. 
     TLC plate  100  described in the above first exemplary embodiment may be configured such that only second layer  32 , among first layer  31  and second layer  32 , has the porous structure modified with a metal oxide. With this configuration, the zeta potential of first layer  31  and the zeta potential of second layer  32  can be different from each other. 
     That is, first layer  31  does not have a metal oxide film. First layer  31  may have a porous structure. The porous structure of first layer  31  may include an aggregate of particles each having a single phase composition, i.e., may be constituted by an aggregate of particles each having a single phase composition. A “particle having a single composition phase” indicates a particle which is uniform in composition. In other words, this means that the particle is not coated with a metal oxide film. 
     Meanwhile, the porous structure of second layer  32  is modified with a metal oxide film. The “porous structure being modified with a metal oxide film” means that the porous structure is coated with a metal oxide film or the surfaces of particles constituting the porous structure are coated with the metal oxide film. That is, second layer  32  has a metal oxide film. The metal oxide film is made of a metal oxide. The metal oxide includes at least one selected from the group consisting of titanium oxide, aluminum oxide, tin oxide, zinc oxide, tungsten oxide, manganese oxide, nickel oxide, copper oxide, and magnesium oxide, for example. The metal oxide may be a semimetal oxide. The semimetal oxide includes at least one selected from the group consisting of boron oxide and silicon dioxide, for example. The material of the metal oxide film is different from the material of the porous structure of second layer  32 . The composition of the metal oxide film is different from the composition of the porous structure of second layer  32 . 
     In TLC plate  100  having the above structure, only second layer  32 , among first layer  31  and second layer  32 , has the porous structure modified with the metal oxide film. Specifically, the interaction between the multiple components included in sample  60  and first layer  31  is different from the interaction between the multiple components and second layer  32 . Therefore, when the multiple components are developed in first layer  31  and second layer  32 , different results can be obtained in first layer  31  and second layer  32 . For example, the multiple components which are not separated from each other in first layer  31  are separated from each other in second layer  32 . The multiple components which are not separated from each other in second layer  32  are separated from each other in first layer  31 . Each of the multiple components can be identified based on the development result of the multiple components in a first stage. Therefore, it is unnecessary to develop the multiple components in a second stage. Thus, sample  60  can be analyzed more easily and more quickly. 
     Second Exemplary Embodiment 
     As shown in  FIGS. 4A and 4B , TLC plate  200  according to the present exemplary embodiment includes separation layer  21  having first layer  31 , second layer  32 , and third layer  33 . The structure of TLC plate  200  is the same as the structure of TLC plate  100  according to the first exemplary embodiment except for third layer  33 . Therefore, constituent elements which are common between TLC plate  100  in the first exemplary embodiment and TLC plate  200  in the present exemplary embodiment are denoted by the same reference marks and may not be described in detail below. That is, the descriptions regarding the following exemplary embodiments are mutually applicable, in so far as they are technically consistent with one another. In addition, the respective exemplary embodiments may be combined with one another, in so far as they are technically consistent with one another. 
     Third layer  33  is a layer having a band shape. Third layer  33  has a rectangular band shape in a plan view. Third layer  33  extends in development direction X. Third layer  33  extends from one of a pair of end faces of substrate  10  to the other in development direction X. Note that third layer  33  may not reach to the other end face of substrate  10 . 
     In the present exemplary embodiment, first layer  31 , second layer  32 , and third layer  33  are disposed on substrate  10 . In other words, first layer  31 , second layer  32 , and third layer  33  are in contact with substrate  10 . First layer  31 , second layer  32 , and third layer  33  are arrayed in this order in array direction Y. Third layer  33  is in contact with second layer  32 . A side surface of third layer  33  and a side surface of second layer  32  are in contact with each other. When separation layer  21  is viewed in a plan view, one side of third layer  33  is in contact with one side of second layer  32 . A length of the one side of third layer  33  is equal to a length of the one side of second layer  32 . Boundary face  41  is formed due to the contact between second layer  32  and third layer  33 . Boundary face  41  extends in development direction X. Note that third layer  33  may not be in contact with second layer  32 . 
     Third layer  33  has a porous structure. The porous structure of third layer  33  can carry the developing solvent from one end to the other end of third layer  33  in development direction X due to capillary force. The material of the porous structure of third layer  33  may be the same as any of those described as examples of the material of the porous structure of first layer  31 . An average pore diameter of the porous structure of third layer  33  may range from 0.01 μm to 100 μm both inclusive. When the porous structure of third layer  33  is formed from an aggregate of inorganic particles or polymer material particles, an average particle diameter of inorganic particles or polymer material particles may range from 1 μm to 100 μm both inclusive. 
     Third layer  33  includes a third metal oxide. The third metal oxide may be the same as any of those described as examples of the first metal oxide. The third metal oxide is different from the material of the porous structure of third layer  33 . The composition of the third metal oxide is different from the composition of the porous structure of third layer  33 . The third metal oxide may be included in the porous structure of third layer  33 . An isoelectric point of the first metal oxide, an isoelectric point of the second metal oxide, and an isoelectric point of the third metal oxide are different from one another. For example, a difference between the isoelectric point of the third metal oxide and the isoelectric point of the first metal oxide and a difference between the isoelectric point of the third metal oxide and the isoelectric point of the second metal oxide are 1 to 8, respectively. 
     The third metal oxide may be in contact with a part of the porous structure of third layer  33 . The third metal oxide may be disposed on the porous structure of third layer  33 . The third metal oxide may be disposed between the porous structure of third layer  33  and substrate  10 . When the porous structure of third layer  33  is formed from an aggregate of inorganic particles or polymer material particles, the third metal oxide may be located between multiple particles constituting the porous structure of third layer  33 . The third metal oxide film may be disposed on the porous structure of third layer  33 . The third metal oxide film is made of the third metal oxide. The third metal oxide film may partially cover the surface of the porous structure of third layer  33 . The third metal oxide film may be disposed between the porous structure of third layer  33  and substrate  10 . The third metal oxide film may partially cover the surface of substrate  10 . When the third metal oxide is in contact with the porous structure of third layer  33 , an interaction between the multiple components included in sample  60  and third layer  33  is accelerated. Thus, the multiple components may be easily separated from each other in third layer  33 . 
     The porous structure of third layer  33  may include an aggregate of particles coated with the third metal oxide film. Third layer  33  may be formed from an aggregate of particles each of which is coated with the third metal oxide film. The particles include at least one kind selected from the group consisting of inorganic particles and polymer material particles, for example. The third metal oxide film may coat the entire surface of the particle or coat a part of the surface of the particle. When the porous structure includes an aggregate of particles coated with the third metal oxide film, the interaction between the multiple components included in sample  60  and third layer  33  is accelerated. Thus, the multiple components may be easily separated from each other in third layer  33 . 
     The thickness of the third metal oxide film is not particularly limited. The thickness of the third metal oxide film is determined according to the material of the third metal oxide film. There is a tendency that, as the third metal oxide film is thicker, the multiple components are more easily separated from each other in third layer  33  when the sample is developed. As the third metal oxide film is thicker, the mobility of the developing solvent in third layer  33  slows down. The thickness of the third metal oxide film ranges from 10 nm to 1000 nm both inclusive, for example. 
     Third layer  33  may further include any of the additives mentioned above. 
     The length of third layer  33  in development direction X is typically equal to length L 1  of first layer  31  of TLC plate  100 . The length of third layer  33  in array direction Y is typically equal to length L 2  of first layer  31  of TLC plate  100 . 
     As a method for forming third layer  33  on substrate  10 , the methods described above as examples of the method for forming first layer  31  and second layer  32  on substrate  10  in the first exemplary embodiment can be used, for example. 
     In TLC plate  200 , the isoelectric point of the first metal oxide, the isoelectric point of the second metal oxide, and the isoelectric point of the third metal oxide are different from one another. Therefore, when the multiple components are developed in first layer  31 , second layer  32 , and third layer  33 , different results can be obtained in first layer  31 , second layer  32 , and third layer  33 . For example, the multiple components which are not separated from each other in first layer  31  and second layer  32  are separated from each other in third layer  33 . Each of the multiple components can be identified based on the development result of the multiple components in a first stage. Therefore, it is unnecessary to develop the multiple components in a second stage. Thus, sample  60  can be analyzed more easily and more quickly. 
     Depending on the multiple components included in sample  60 , third layer  33  may not include the third metal oxide. In such a case, TLC plate  200  needs to satisfy at least one requirement selected from among the requirement in which first layer  31 , second layer  32 , and third layer  33  are different in composition and the requirement in which first layer  31 , second layer  32 , and third layer  33  are different in structure. When the above requirement is satisfied, first layer  31 , second layer  32 , and third layer  33  induce different interactions with the multiple components included in sample  60 . Therefore, when the multiple components are developed in first layer  31 , second layer  32 , and third layer  33 , different results can be obtained in first layer  31 , second layer  32 , and third layer  33 . “First layer  31 , second layer  32 , and third layer  33  being different in structure” means that first layer  31 , second layer  32 , and third layer  33  are different in at least one factor selected from among an average pore diameter of the porous structure, a void ratio of the porous structure, and an average particle diameter of the material of the porous structure, for example. 
     TLC plate  200  described in the above second exemplary embodiment may be configured such that first layer  31  does not have a metal oxide film as in the first exemplary embodiment. With this configuration, the effect same as the effect of the first exemplary embodiment can be obtained. 
     Third Exemplary Embodiment 
     As shown in  FIGS. 5A and 5B , TLC plate  300  according to the present exemplary embodiment is obtained by further providing fourth layer  34  to n-th layer  35  to the configuration of TLC plate  200  in the second exemplary embodiment. Each of fourth layer  34  to n-th layer  35  induces an interaction different from the interactions induced by first layer  31 , second layer  32 , and third layer  33 , with respect to multiple components included in sample  60 . Therefore, when the multiple components are developed in first layer  31  to n-th layer  35 , different results can be obtained in first layer  31  to n-th layer  35 . For example, the multiple components which are not separated from each other in first layer  31 , second layer  32 , and third layer  33  are separated from each other in any one of fourth layer  34  to n-th layer  35 . 
     Each of fourth layer  34  to n-th layer  35  is a layer having a band shape. Each of fourth layer  34  to n-th layer  35  has a rectangular band shape in a plan view. n is an integer equal to or greater than 4. n is an integer from 5 to 10, for example. Each of fourth layer  34  to n-th layer  35  extends in development direction X. Each of fourth layer  34  to n-th layer  35  extends from one of a pair of end faces of substrate  10  to the other in development direction X. Note that each of fourth layer  34  to n-th layer  35  may not reach to the other end face of substrate  10 . 
     In the present exemplary embodiment, first layer  31  to n-th layer  35  are disposed on substrate  10 . In other words, first layer  31  to n-th layer  35  are in contact with substrate  10 . First layer  31  to n-th layer  35  are arrayed in this order in array direction Y. Each of fourth layer  34  to n-th layer  35  is in contact with the corresponding one of third layer  33  to (n−1)th layer (not shown). When separation layer  22  is viewed in a plan view, one side of each of fourth layer  34  to n-th layer  35  is in contact with one side of the corresponding one of third layer  33  to (n−1)th layer. A length of the one side of each of fourth layer  34  to n-th layer  35  is equal to a length of the one side of the corresponding one of third layer  33  to (n−1)th layer. Note that each of fourth layer  34  to n-th layer  35  may not be in contact with the corresponding one of third layer  33  to (n−1)th layer. 
     Each of fourth layer  34  to n-th layer  35  has a porous structure. The porous structure of each of fourth layer  34  to n-th layer  35  can carry the developing solvent from one end to the other end of each of fourth layer  34  to n-th layer  35  in development direction X due to capillary force. The material of the porous structure of each of fourth layer  34  to n-th layer  35  may be the same as any of those described above as examples of the porous structure of first layer  31 . An average pore diameter of the porous structure of each of fourth layer  34  to n-th layer  35  may range from 0.01 μm to 100 μm both inclusive. When the porous structure of each of fourth layer  34  to n-th layer  35  is formed from an aggregate of inorganic particles or polymer material particles, an average particle diameter of inorganic particles or polymer material particles may range from 1 μm to 100 μm both inclusive. 
     Each of fourth layer  34  to n-th layer  35  includes a corresponding one of a fourth metal oxide to an n-th metal oxide. Each of the fourth metal oxide to the n-th metal oxide may be the same as any of those described as examples of the first metal oxide. Each of the fourth metal oxide to the n-th metal oxide is different from the material of the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 . The composition of each of the fourth metal oxide to the n-th metal oxide is different from the composition of the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 . Each of the fourth metal oxide to the n-th metal oxide may be included in the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 . The first metal oxide to the n-th metal oxide are different in isoelectric point. 
     Each of the fourth metal oxide to the n-th metal oxide may be in contact with a part of the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 . Each of the fourth metal oxide to the n-th metal oxide may be disposed on the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 . Each of the fourth metal oxide to the n-th metal oxide may be disposed between the porous structure of the corresponding one of fourth layer  34  to n-th layer  35  and substrate  10 . When the porous structures of fourth layer  34  to n-th layer  35  are formed from an aggregate of inorganic particles or polymer material particles, each of the fourth metal oxide to the n-th metal oxide may be located between multiple particles constituting the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 . Each of the fourth metal oxide film to the n-th metal oxide film may be disposed on the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 . Each of the fourth metal oxide film to the n-th metal oxide film is made of the corresponding one of the fourth metal oxide to the n-th metal oxide. Each of the fourth metal oxide film to the n-th metal oxide film may partially cover the surface of the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 . Each of the fourth metal oxide film to the n-th metal oxide film may be disposed between substrate  10  and the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 . Each of the fourth metal oxide film to the n-th metal oxide film may partially cover the surface of substrate  10 . When each of the fourth metal oxide to the n-th metal oxide is in contact with the porous structure of the corresponding one of fourth layer  34  to n-th layer  35 , the interaction between the multiple components included in sample  60  and each of fourth layer  34  to n-th layer  35  is accelerated. Thus, the multiple components may be easily separated from each other in each of fourth layer  34  to n-th layer  35 . 
     The porous structure of each of fourth layer  34  to n-th layer  35  may include an aggregate of particles coated with the corresponding one of the fourth metal oxide film to the n-th metal oxide film. Each of fourth layer  34  to n-th layer  35  may be constituted by an aggregate of particles each of which is coated with the corresponding one of the fourth metal oxide film to the n-th metal oxide film. The particles include at least one kind selected from the group consisting of inorganic particles and polymer material particles, for example. The fourth metal oxide film to the n-th metal oxide film may coat the entire surfaces of the particles or coat a part of the surfaces of the particles. When the porous structure of each of fourth layer  34  to n-th layer  35  includes an aggregate of particles coated with the corresponding one of the fourth metal oxide film to the n-th metal oxide film, the interaction between the multiple components included in sample  60  and each of fourth layer  34  to n-th layer  35  is accelerated. Thus, the multiple components may be easily separated from each other in each of fourth layer  34  to n-th layer  35 . 
     The thickness of each of the fourth metal oxide film to the n-th metal oxide film is not particularly limited. The thickness of each of the fourth metal oxide film to the n-th metal oxide film is determined according to the material of the corresponding one of the fourth metal oxide film to the n-th metal oxide film. There is a tendency that, as each of the fourth metal oxide film to the n-th metal oxide film is thicker, the multiple components are more easily separated from each other when the sample is developed in development direction X. As each of the fourth metal oxide film to the n-th metal oxide film is thicker, the mobility of the developing solvent in each of fourth layer  34  to n-th layer  35  slows down. The thickness of each of the fourth metal oxide film to the n-th metal oxide film ranges from 10 nm to 1000 nm both inclusive, for example. 
     Fourth layer  34  to n-th layer  35  may further include any of the additives mentioned above. 
     The length of each of fourth layer  34  to n-th layer  35  in development direction X is typically equal to length L 1  of first layer  31  of TLC plate  100 . The length of each of fourth layer  34  to n-th layer  35  in array direction Y is typically equal to length L 2  of first layer  31  of TLC plate  100 . 
     As a method for forming each of fourth layer  34  to n-th layer  35  on substrate  10 , the methods described above as examples of the method for forming first layer  31  and second layer  32  on substrate  10  in the first exemplary embodiment can be used, for example. 
     In TLC plate  300 , the first metal oxide to the n-th metal oxide are different in isoelectric point. Therefore, when the multiple components are developed in first layer  31  to n-th layer  35 , different results can be obtained in first layer  31  to n-th layer  35 . For example, the multiple components which are not separated from each other in first layer  31 , second layer  32 , and third layer  33  are separated from each other in any one of fourth layer  34  to n-th layer  35 . Each of the multiple components can be identified based on the development result of the multiple components in a first stage. Therefore, it is unnecessary to develop the multiple components in a second stage. Thus, sample  60  can be analyzed more easily and more quickly. 
     Depending on the multiple components included in sample  60 , each of fourth layer  34  to n-th layer  35  may not include the corresponding one of the fourth metal oxide to the n-th metal oxide. In such a case, TLC plate  300  needs to satisfy at least one requirement selected from among the requirement in which first layer  31  to n-th layer  35  are different in composition and the requirement in which first layer  31  to n-th layer  35  are different in structure. When the above requirement is satisfied, first layer  31  to n-th layer  35  induce different interactions with the multiple components included in sample  60 . Therefore, when the multiple components are developed in first layer  31  to n-th layer  35 , different results can be obtained in first layer  31  to n-th layer  35 . “First layer  31  to n-th layer  35  being different in structure” means that first layer  31  to n-th layer  35  are different in at least one factor selected from among an average pore diameter of the porous structure, a void ratio of the porous structure, and an average particle diameter of the material of the porous structure, for example. 
     TLC plate  300  described in the above third exemplary embodiment may be configured such that first layer  31  does not have a metal oxide film as in the first exemplary embodiment. With this configuration, the effect same as the effect of the first exemplary embodiment can be obtained. 
     Fourth Exemplary Embodiment 
     As shown in  FIGS. 6A and 6B , TLC plate  400  according to the present exemplary embodiment includes functional layer  50  disposed on separation layer  20 . The structure of TLC plate  400  is the same as the structure of TLC plate  100  in the first exemplary embodiment except for functional layer  50 . When sample  60  is placed on functional layer  50 , sample  60  penetrates into functional layer  50 . Sample  60  spreads all over functional layer  50 . Sample  60  penetrating into functional layer  50  is brought into contact with separation layer  20 . Therefore, it is unnecessary to place sample  60  on separation layer  20  several times. Thus, sample  60  can be efficiently placed on separation layer  20 . 
     Functional layer  50  is a layer having a band shape. Functional layer  50  has a rectangular band shape in a plan view. Functional layer  50  is in contact with first layer  31  and second layer  32 . Functional layer  50  extends in array direction Y. Functional layer  50  extends from one of a pair of end faces of substrate  10  to the other in array direction Y. Note that functional layer  50  may not extend from the one end face of substrate  10 , as long as it is in contact with first layer  31  and second layer  32 . Functional layer  50  may not reach to the other end face of substrate  10 . 
     Functional layer  50  is disposed on first layer  31  and second layer  32 . A lower surface of functional layer  50  and an upper surface of first layer  31  constitute boundary face  42 . The lower surface of functional layer  50  and an upper surface of second layer  32  constitute boundary face  43 . Boundary faces  42  and  43  extend in array direction Y. 
     Functional layer  50  has a porous structure. The material of the porous structure of functional layer  50  may be the same as any of those described as examples of the porous structure of first layer  31 . An average pore diameter of the porous structure of functional layer  50  may range from 0.01 μm to 100 μm both inclusive. When the porous structure of functional layer  50  is formed from an aggregate of inorganic particles or polymer material particles, an average particle diameter of inorganic particles or polymer material particles may range from 1 μm to 100 μm both inclusive. Functional layer  50  may further include any of the additives mentioned above. 
     The distance from end  31   a  of first layer  31  to functional layer  50  in development direction X is determined according to the liquid level of developing solvent  70 , for example. The length of functional layer  50  in development direction X is determined according to an amount of sample  60  to be placed on functional layer  50 , for example. The thickness of functional layer  50  is determined according to the porous structure of functional layer  50 , for example. The thickness of functional layer  50  is typically equal to thickness L 4  of first layer  31 . 
     As a method for forming functional layer  50  on separation layer  20 , the methods described above as examples of the method for forming first layer  31  and second layer  32  on substrate  10  in the first exemplary embodiment can be used, for example. 
     Functional layer  50  has a porous structure. Therefore, when sample  60  is placed on functional layer  50 , sample  60  penetrates into functional layer  50 . Sample  60  spreads all over functional layer  50 . Sample  60  penetrating into functional layer  50  is brought into contact with separation layer  20 . Specifically, sample  60  penetrating into functional layer  50  is brought into contact with first layer  31  via boundary face  42 . Thus, sample  60  penetrates into first layer  31 . Sample  60  penetrating into functional layer  50  is brought into contact with second layer  32  via boundary face  43 . Thus, sample  60  penetrates into second layer  32 . Since sample  60  spreads all over functional layer  50 , it is unnecessary to place sample  60  on separation layer  20  several times. Thus, sample  60  can be efficiently placed on separation layer  20 . The volume of sample  60  to be placed on functional layer  50  ranges from 2 μL to 20 μL both inclusive, for example. 
     TLC plate  400  described in the above fourth exemplary embodiment may be configured such that first layer  31  does not have a metal oxide film as in the first exemplary embodiment. With this configuration, the effect same as the effect of the first exemplary embodiment can be obtained. 
     INDUSTRIAL APPLICABILITY 
     The technique disclosed in the present specification is useful for protein analysis or the like. 
     REFERENCE MARKS IN THE DRAWINGS 
     
         
         
           
               10 : substrate 
               20 ,  21 ,  22 : separation layer 
               31 : first layer 
               32 : second layer 
               50 : functional layer 
               60 : sample 
               100 ,  200 ,  300 ,  400 : TLC plate (thin layer chromatography plate) 
             X: development direction (first direction) 
             y: array direction (second direction)