WO2013005642A1 - スポット検出用セット、スポット検出方法、及び被転写シート - Google Patents
スポット検出用セット、スポット検出方法、及び被転写シート Download PDFInfo
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- WO2013005642A1 WO2013005642A1 PCT/JP2012/066563 JP2012066563W WO2013005642A1 WO 2013005642 A1 WO2013005642 A1 WO 2013005642A1 JP 2012066563 W JP2012066563 W JP 2012066563W WO 2013005642 A1 WO2013005642 A1 WO 2013005642A1
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- separating agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/95—Detectors specially adapted therefor; Signal analysis
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- the present invention relates to a spot detection set for detecting a spot of a target substance in a separating agent layer having no optical response on a TLC plate, a spot detection method using the spot detection set, and a transfer sheet in the set.
- TLC thin layer chromatography
- TLC is used for detection and fractionation of a target substance in a sample.
- a spot of a target substance is usually detected by a difference in optical response between the separating agent layer of the TLC plate and the target substance. Therefore, in TLC, when the optical response of the separating agent layer and the target substance is the same, the spot may not be detected by the optical response.
- a TLC plate in which two separating agent layers are formed side by side is known (for example, see Patent Document 1).
- the target substance in the sample is developed from the first separating agent layer to the second separating agent layer, and the spot separated by the first separating agent layer moves to the adjacent second separating agent layer. Therefore, it is detected according to the optical response.
- the extract component that is easily adsorbed by the first separating agent layer in the sample may not reach the second separating agent layer sufficiently.
- the positional relationship of the spots in the first separating agent layer may not be accurately maintained up to the second separating agent layer.
- the TLC plate may not be able to accurately detect the separation state in the first separating agent layer, and at least room for examination is left in this respect.
- the present invention provides a technique for accurately detecting a spot on a TLC plate in which a target substance cannot be detected by optical response.
- the present inventors have found that the above-mentioned problems can be solved by transferring the spot on the TLC plate to a transfer sheet having optical response to the target substance, and have completed the present invention.
- the present invention has a TLC plate having a separating agent layer, and a transfer sheet on which the spot of the separating agent layer is transferred by being superimposed on the separating agent layer, and the separating agent layer is separable from the target substance. And a spot detection set in which the transfer sheet has optical response and porosity different from those of the separating agent layer.
- the present invention also provides the spot detection set as described above, wherein the transfer sheet is a silica gel sheet.
- the present invention also provides the above spot detection set further comprising an alignment structure for overlapping the TLC plate and the transfer sheet in a predetermined positional relationship.
- the present invention also relates to a method for detecting a spot in thin layer chromatography using the spot detection set of the present invention described above, comprising the steps of developing a target substance in a sample on a separating agent layer of a TLC plate; A step of superimposing a transfer sheet on a position where at least a target substance is developed in the agent layer, a step of wetting the overlapped separation agent layer and the transfer sheet with a transfer solvent, and an overlap of the separation agent layer.
- a spot detection method comprising: a step of volatilizing a transfer solvent from a transfer sheet side to transfer a spot of a separating agent layer to the transfer sheet; and a step of optically detecting the spot transferred to the transfer sheet.
- the present invention also provides the spot detection method described above, further comprising a step of drying the separating agent layer in which the target substance is developed before the transfer sheet is overlaid on the separating agent layer.
- the present invention also provides the spot detection method described above in which the transfer target sheets are superposed in a predetermined positional relationship with respect to the TLC plate.
- the present invention provides a separation agent for use in transferring a spot of a separation agent layer from a separation agent layer in a TLC plate provided with a separation agent layer having a separation property for a target substance and an optical response property to ultraviolet light or a coloring reagent.
- a separation agent for use in transferring a spot of a separation agent layer from a separation agent layer in a TLC plate provided with a separation agent layer having a separation property for a target substance and an optical response property to ultraviolet light or a coloring reagent.
- a sheet to be transferred having optical response and porosity different from those of an agent layer.
- the present invention also provides the above-mentioned transferred sheet which is a silica gel sheet.
- the present invention uses the transfer sheet superimposed on the separation agent layer of the TLC plate, by volatilizing the transfer solvent from the back surface of the transfer sheet superimposed on the separation agent layer, Since at least a part of the spot is transferred to the transfer sheet, it is possible to accurately detect the spot on the TLC plate where the target substance cannot be detected by optical response.
- FIG. It is a figure which shows an example of the spot detection method of this invention. It is a figure which shows the spot of the Tregger base in the TLC plate 1 which has a 1st and 2nd separating agent layer. It is a figure which shows the spot of flavanone in the TLC plate 1.
- FIG. It is a figure which shows the spot of the tregar base and flavanone in the to-be-transferred sheet which transcribe
- the spot detection set of the present invention includes a TLC plate having a separating agent layer and a transfer sheet on which the spot of the separating agent layer is transferred while being superimposed on the separating agent layer.
- the TLC plate is not particularly limited as long as it is a TLC plate having a separating agent layer having separability with respect to a target substance and optical responsiveness with respect to ultraviolet rays or a coloring reagent.
- the optical responsiveness to ultraviolet rays refers to light emission or absorption of ultraviolet rays such as fluorescence.
- the optical responsiveness to the coloring reagent refers to color development by the coloring reagent.
- An example of such a TLC plate is a TLC plate having a substrate and the separating agent layer formed on the substrate.
- Such a TLC plate can be appropriately selected from known TLC plates, and the separation agent slurry having the above-mentioned separation property and optical response is applied on a substrate and dried to form a separation agent layer. Can be obtained.
- a known substrate in TLC can be used.
- examples of such a substrate include a flat plate made of glass, resin, metal, or paper.
- substrate is not specifically limited, It is preferable that it is a rectangle normally used by TLC.
- a particulate separating agent can be used as the separating agent.
- a particulate separating agent include particles composed only of the separating agent, and particles obtained by supporting the separating agent on a particulate carrier.
- the separating agent either a low molecular separating agent or a high molecular separating agent having the optical response can be used.
- the low molecular separation agent include a ligand exchange type separation agent, a charge transfer ( ⁇ - ⁇ ) type separation agent, a hydrogen bond type separation agent, an inclusion type separation agent, and an ion bond type separation agent.
- examples thereof include a separating agent, an intercalating type separating agent, a crown ether or a derivative thereof, and a cyclodextrin or a derivative thereof.
- the polymer separating agent include polysaccharide derivatives, polyamides, polymethacrylic acid esters, polyacrylamides, proteins, and tartaric acid derivatives.
- polysaccharide derivatives examples include aromatic ester groups, aromatic carbamoyl groups, aromatic ether groups, and carbonyls, which are used in separating agents for optical isomers, for example, by replacing part or all of hydroxyl groups or amino groups of polysaccharides and polysaccharides.
- examples thereof include polysaccharide derivatives composed of any of the groups, and examples thereof include phenyl carbamate derivatives of cellulose, phenyl ester derivatives of cellulose, phenyl carbamate derivatives of amylose, and phenyl ester derivatives of amylose.
- the phenyl group in these derivatives may have one or more substituents selected from the group consisting of hydrocarbons having 1 to 20 carbon atoms and halogens.
- the carrier is preferably a porous body from the viewpoint of improving the separation performance.
- the carrier include synthetic polymers such as cross-linked polystyrene, cross-linked acrylic polymer, and epoxy polymer, cellulose and cross-linked cellulose reinforced by cross-linking, cross-linked agarose, cross-linked dextran, and cross-linked mannan cross-linked polysaccharides, and , Alumina, silica gel, mesoporous silica gel, zeolite, diatomaceous earth, fused silica, viscosity mineral, zirconia, metals and other inorganic materials.
- the particle size of the separating agent can be determined according to the purpose of separation in the TLC plate. For example, if the conditions for medium pressure column chromatography are to be examined, from the viewpoint of obtaining results applicable to medium pressure chromatography, The thickness is preferably 10 ⁇ m or more, more preferably 10 to 100 ⁇ m, and further preferably 20 to 100 ⁇ m.
- an average particle size measured by a normal particle size measuring device can be adopted, but it may be a catalog value.
- the separating agent layer is formed using a known method for producing a TLC plate, for example, a slurry containing the separating agent and a coating solvent, and a surface of a support using a spreader. It can be formed by applying to the surface or by spraying the slurry onto the surface of the support.
- the coating solvent water, an organic solvent, and a mixed solvent thereof can be used.
- the organic solvent include alcohols such as methanol, ethanol and isopropanol, ketones such as acetone and ethyl methyl ketone, ethers such as tetrahydrofuran and dioxane, nitriles such as acetonitrile, sulfoxides such as dimethyl sulfoxide, sulfones such as sulfolane, and the like.
- Esters such as ethyl acetate, amides such as dimethylformamide, hydrocarbons such as pentane, hexane, petroleum ether, aromatic hydrocarbons such as benzene, toluene, xylene, methylene chloride, chloroform, bromoform, chlorobenzene, And halogen-containing compounds such as bromobenzene.
- the coating solvent is preferably a mixed solvent of a water-soluble organic solvent and water, more preferably a mixed solvent of alcohol and water, and further preferably a mixed solvent of ethanol and water.
- the alcohol content in the mixed solvent is preferably 0.1 to 50% by mass, more preferably 10 to 40% by mass, and further preferably 20 to 30% by mass.
- the type of the coating solvent is preferably the same, and the composition of the coating solvent is more preferably the same.
- the content of the coating solvent in the slurry can be determined from the uniformity of the separating agent layer to be formed, the thickness of the layer, and an economical point of view.
- the amount is preferably 5,000 parts by mass, more preferably 50 to 1,000 parts by mass, and still more preferably 100 to 300 parts by mass.
- the slurry preferably further contains a binder from the viewpoint of improving the strength of the separating agent layer to be formed.
- a binder a component capable of binding to form a separating agent layer on the surface of the substrate can be used.
- binders include inorganic binders such as gypsum and colloidal silica, organic fibers such as microfibrillated cellulose, alkali water-soluble copolymers, thickeners such as hydroxyethyl cellulose and carboxymethyl cellulose, polyvinyl alcohol, acrylic
- An organic binder such as an acid may be used.
- the binder may be one type or two or more types.
- the content of the binder in the slurry can be appropriately determined according to the type of the binder from the viewpoint of the strength of the separating agent layer to be formed and the appropriate rising speed of the mobile phase in the separating agent layer.
- the content of the binder is preferably 0.1 to 50 parts by mass, more preferably 5 to 30 parts by mass with respect to 100 parts by mass of the separating agent, and 10 to 20 parts by mass. More preferably.
- the content of the binder is preferably 0.1 to 50 parts by mass with respect to 100 parts by mass of the separating agent, and 0.5 to 10 parts by mass. More preferably, it is 1 to 3 parts by mass.
- the transfer sheet has optical response and porosity different from the separating agent layer.
- “different optical responsiveness” means that one optical response and the other optical response due to ultraviolet irradiation or color development treatment of a coloring reagent are different from each other to the extent that they can be optically discriminated by color and brightness. To tell.
- “Porosity” of the transfer sheet means that when the transfer sheet is superimposed on the separating agent layer, both are wetted with a solvent and dried from the transfer sheet side, a spot of the separating agent layer is formed. It means that there is a gap through which the solvent is sucked onto the transfer sheet at such a speed that at least a part of the components are transferred to the transfer sheet.
- the porosity (total porosity) of the transferred sheet is preferably 0.1 to 0.9, more preferably 0.2 to 0.8, and 0.4 to 0. More preferably, it is .8.
- the void ratio of the transfer sheet is calculated from, for example, the weight difference obtained by subtracting the weight of the transfer sheet from the weight of the transfer sheet in which the solvent is sucked up, the specific gravity of the sucked solvent, and the outer dimension of the transfer sheet. It can be calculated from the volume to be obtained.
- the porosity of the transfer sheet is preferably uniform (for example, the porosity is constant) in the portion of the transfer sheet that overlaps the TLC plate from the viewpoint of moving the component that forms the spot from the TLC plate. More preferably, it is uniform throughout the plate.
- the transfer sheet can be produced by various methods.
- the transfer sheet can be prepared by applying a slurry of the carrier such as silica gel to a porous support and drying it.
- the porous support include a plate provided with holes by punching, a sintered plate, a mesh (net), and a porous film.
- the material for the support include glass, plastic, metal, and ceramic.
- the transfer sheet contains the carrier and the coating solvent, and if necessary, a slurry containing the binder and aggregate is applied onto the substrate and dried to peel off the layer formed from the substrate.
- the aggregate include a mesh and a filler (fiber).
- the binder content in the slurry for the transferred sheet is preferably 0.1 to 50 parts by weight, and 0.5 to 30 parts by weight with respect to 100 parts by weight of the carrier. More preferred is 1 to 20 parts by mass.
- the aggregate content in the transfer sheet slurry is preferably 0.1 to 0.9 parts by mass with respect to 100 parts by mass of the carrier from the viewpoint of the strength of the transfer sheet. The amount is more preferably 2 to 0.8 parts by mass, and further preferably 0.3 to 0.7 parts by mass.
- the transfer sheet can be produced as porous silica gel (monolith plate) formed into a sheet by a sol-gel method.
- the transfer-receiving sheet is a silica gel sheet in which silica gel is formed into a sheet shape by any of the methods described above. It is preferable from the viewpoint of affinity.
- the thickness of the transferred sheet is preferably 10 to 2000 ⁇ m, more preferably 50 to 1000 ⁇ m, from the viewpoints of permeability of the target substance, prevention of spot diffusion of the target substance and strength of the transferred sheet, More preferably, the thickness is 100 to 500 ⁇ m.
- the particle size of the carrier is: The thickness is preferably 0.1 to 100 ⁇ m, more preferably 1 to 50 ⁇ m, still more preferably 5 to 40 ⁇ m.
- the transfer sheet detects a spot by optical response due to ultraviolet irradiation
- the transfer sheet further contains a fluorescent indicator from the viewpoint of clearer detection.
- a fluorescent indicator a known fluorescent indicator can be used, and examples thereof include magnesium tungstate and manganese-containing zinc silicate.
- Such a transferred sheet can be obtained by further adding a fluorescent indicator to the aforementioned transferred sheet slurry.
- the content of the fluorescent indicator can be determined within a range in which the target substance can be separated, and is generally 0.1 to 5 parts by mass with respect to 100 parts by mass of the carrier.
- a known technique of the coloring reagent and its coloring process can be adopted for the transfer sheet.
- a coloring reagent include anisaldehyde solution, phosphomolybdic acid solution, iodine, ninhydrin solution, chameleon solution, DNPH solution, manganese chloride solution, and bromocresol green solution.
- the coloring process include a process in which a coloring reagent is applied to the transfer sheet by application, dispersion, or exposure, and the transfer sheet is heated as necessary to develop a color.
- the spot detection set of the present invention preferably further has an alignment structure for superimposing the TLC plate and the transfer sheet in a predetermined positional relationship from the viewpoint of more accurate detection of spots in the separating agent layer.
- an alignment structure for example, a position in which the two overlap each other is specified, and a structure in which the overlapping both are prevented from shifting in the horizontal direction can be mentioned, and more specifically, a TLC plate and a transfer sheet are included. With a hinge at the end, a frame that accommodates both the TLC plate and the transferred sheet superimposed on it in the horizontal direction without gaps, a pile provided on one of the TLC plate and the transferred sheet, etc.
- a concave portion such as a hole provided on the other side corresponding thereto and an adhesive means for adhering or detachably attaching a TLC plate such as an adhesive tape or a hook-and-loop fastener to a transfer sheet.
- the spot detection method of the present invention can be performed using the aforementioned spot detection set of the present invention.
- the spot detection method of the present invention includes a first step of developing a target substance in a sample on a separating agent layer of a TLC plate, and a second step of superimposing a transfer sheet on a position where at least the target substance of the separating agent layer is developed.
- the third step of wetting the separated separating agent layer and the transferred sheet with the transfer solvent, and the separating agent by volatilizing the transferring solvent from the transferred sheet side superimposed on the separating agent layer.
- the first step can be performed in the same manner as normal TLC. That is, in the first step, the sample is spotted on one end of the separating agent layer, the spot of the sample is dried if necessary, and immersed in the mobile phase with one end of the separating agent layer on which the sample is spotted facing down.
- the separation can be performed by developing the target substance in the sample in the separating agent layer.
- the target substance is a substance that exhibits the same optical response as that of the separating agent layer in the separating agent layer.
- the transfer sheet may be superposed at a position where the target substance is developed in the separation agent layer, and may be overlaid only on a part of the separation agent layer such as a portion where the mobile phase is developed. In order to detect the position of the spot, it is preferable that the layer is superposed on the entire separating agent layer.
- the second step is preferably a step of overlapping the transfer sheet with a predetermined positional relationship with respect to the TLC plate from the viewpoint of more accurately detecting the position of the spot in the separating agent layer.
- the transfer sheet and the separating agent layer that are superimposed are sprayed onto the entire transfer sheet from the transfer sheet side, or are immersed in the transfer solvent, or This can be done by wetting the transfer sheet with a transfer solvent before superposition so that the separating agent layer is sufficiently wet when superposed.
- a solvent that elutes at least a part of the components forming the spot of the separating agent layer from the separating agent layer can be used.
- a transfer solvent may be the above-mentioned mobile phase, a known organic solvent, or two or more selected from the group consisting of a known organic solvent, water, acid, and alkali. It may be a mixture.
- alcohols such as ethanol, methanol and 2-propanol
- solvents such as THF, ethyl acetate, chloroform and acetone can be used.
- the separating agent layer is prepared using a coating type in which the polysaccharide derivative-based separating agent is supported on the carrier, the polysaccharide derivative-based separating agent supported on the carrier is not dissolved. It is preferable to use alcohols.
- the fourth step is preferably air-dried from the side of the transfer sheet superimposed on the separating agent layer from the viewpoint of sucking up the transfer solvent at an appropriate sucking speed on the transfer-receiving sheet, and is air-dried by cold air at 10 to 30 ° C. Further, it is preferable from the viewpoint of increasing the transfer amount. This air drying may be performed on the entire transferred plate, or may be performed on a portion of the transferred plate such as a portion where a spot will be present.
- the fifth step may be performed while the transfer sheet is superimposed on the separating agent plate, or may be performed after the transfer sheet is peeled off.
- the target substance exhibits an optical response that is distinguishably different from the optical response of the transfer sheet.
- the optical detection in the fifth step is performed on the front side of the transfer sheet within the range where the optical response of the transferred spot is obtained, when the contact surface of the transfer sheet with the separating agent plate is the front side. It may be performed on the back side.
- the spot detection method of the present invention may include further steps as long as the effects of the present invention are obtained.
- Examples of such other steps include a step of drying the separating agent layer in which the target substance is developed before superimposing the transfer sheet on the separating agent layer. Further including this drying step is preferable from the viewpoint of more accurately detecting the position of the spot in the separating agent layer.
- This drying step is preferably a step of air drying with hot air at 20 to 80 ° C. from the viewpoint of improving workability, and a step of air drying with cold air is preferable from the viewpoint of safety.
- a TLC plate having a separating agent layer made of silica gel coated with a polysaccharide derivative is prepared (A in the figure), and one end side of the separating agent layer of this TLC plate is prepared.
- a sample such as a racemic solution of the optical isomer is spotted on the sample, the optical isomer in the sample is spread from one end side to the other end side, and once dried with cold air by a dryer (B in the figure)
- a transfer sheet such as a sheet made of silica gel is overlaid on the separating agent layer of the TLC plate (C in the figure), and a transfer solvent such as ethanol is sprayed on the entire back surface of the overlapped transfer sheet to obtain the transfer sheet.
- Both of the separating agent layers are wetted with a transfer solvent (D in the figure), and both of the superimposed transfer sheet and separating agent layer are gradually dried from the back side of the transferred sheet with cold air from a dryer (in the figure). ), By irradiating UV on the back side of the transfer sheet, it can be performed by detecting a spot that has been transferred to the transfer sheet optically (F in the figure).
- the optical detection of spots on the transfer sheet in the present invention can be used for observing the separation results on the TLC plate.
- both the spot of the raffinate component and the extract component can be accurately detected including their positional relationship by separation by TLC.
- by collecting a portion including a specific spot transferred from the transfer sheet and performing an extraction operation it can also be used for fractionation of a target substance.
- the part of the separating agent layer corresponding to the part including the specific spot detected on the transfer sheet can also be collected and used for the separation of the target substance.
- silica gel for liquid chromatography manufactured by Daiso Corporation, IR-60-5 / 20-U
- 0.20 g of gypsum, 2% CMC (carboxymethylcellulose) 1110 manufactured by Daicel Chemical Industries, Ltd.
- aqueous solution 6 0.002 g and manganese-containing zinc silicate 0.04 g were added to a mixed solution of water 2.02 g and ethanol 2.80 g, and stirred well while irradiating ultrasonic waves to prepare a second slurry.
- These slurries are uniformly applied to the surface of the glass plate using a spreader for preparing a TLC plate equipped with a partition plate.
- Each of the applied slurry layers is air-dried and vacuum-dried at 60 ° C. for 3 hours while being drawn with a vacuum pump. By doing so, 12 TLC plates 1 having the first separating agent layer by the first slurry and the second separating agent layer attached to the first separating agent layer by the second slurry were formed. .
- the first separating agent layer has a width of 2 cm, a length of 10 cm and a thickness of 150 ⁇ m, and the second separating agent layer has a width of 3 cm, a length of 10 cm and a thickness of 110 ⁇ m.
- the first separating agent layer is a layer made of IA filler, and the second separating agent layer is the silica gel layer.
- the average particle diameter of the IA filler is 20 ⁇ m, and the average particle diameter of the silica gel is 14.4 ⁇ m.
- TLC plate 1 About 1 ⁇ L of 1% ethyl acetate solution of racer base (TB) racemate was spotted at a position about 1.0 cm from the bottom when the longitudinal direction of the TLC plate 1 was vertical.
- the TLC plate 1 is accommodated in a developing tank containing a mixed solvent containing n-hexane and ethanol at a volume ratio of 9: 1 with the sample spot facing down, and the sample is placed in the longitudinal direction of the first separating agent layer.
- the optical isomers of Tregar base in the middle were developed. After this first development, the TLC plate 1 is once dried with cold air, and then the TLC plate 1 is accommodated in the development tank containing ethanol so that the first separating agent layer is located below.
- the optical isomer of Tregar base was further developed from the agent layer to the second separating agent layer.
- the TLC plate 1 was dried with cold air, and the TLC plate 1 was irradiated with ultraviolet rays.
- the raffinate component RTB and the extract component moved from the first separating agent layer to the second separating agent layer.
- ETB spots were confirmed as green spots in the second separating agent layer (FIG. 2).
- the first separating agent layer was observed as a black region by absorbing ultraviolet rays.
- the Rf value of each spot was determined from the spotting position of the sample in the first separating agent layer, the arrival position of the developing solution, and the center position of the spot in the second separating agent layer.
- the Rf value was about 0.5
- the Rf value of the extract component was about 0.35.
- the first slurry is uniformly applied to the surface of the glass plate using a spreader for preparing a TLC plate, and the applied slurry layer is air-dried and vacuum-dried at 60 ° C. for 3 hours while being drawn with a vacuum pump. 2 cm, length 10 cm, and thickness 150 ⁇ m, the TLC plate 2 having only the first separating agent layer made of the first slurry was formed.
- TLC plate 2 is housed in a developing tank containing a mixed solvent containing n-hexane and ethanol at a volume ratio of 9: 1, respectively, with the spot of both samples facing down, spotted at a position of about 1.0 cm. Both samples were developed simultaneously in the longitudinal direction of the TLC plate 2. After this development, the TLC plate 2 was once dried with cold air to stop the development of the spots.
- a silica gel sheet formed in a rectangular shape of the same size is superposed, and ethanol is sprayed on the entire laminated silica gel sheet so that the silica gel sheet and the first separating agent are sprayed.
- the spot of the separating agent layer was transferred from the first separating agent layer to a silica gel sheet.
- the silica gel sheet was prepared by applying the second slurry onto the glass plate as described above and drying the second separating agent layer having a thickness of about 300 ⁇ m from the glass plate to form the rectangle. It was formed by adjusting the shape.
- the silica gel sheet was removed from the TLC plate 2 and the contact surface of the silica gel sheet with the TLC plate 2 was irradiated with ultraviolet rays. Each was detected as a green spot (FIG. 4).
- the third slurry is the same as the first slurry except that a filler of CHIRALPAK IC (registered trademark of the company) manufactured by Daicel Chemical Industries, Ltd. (also referred to as “IC filler”) is used instead of the IA filler.
- IC filler also referred to as “IC filler”.
- a TLC plate 3 having only a third separating agent layer of the third slurry was formed in the same manner as the TLC plate 2 except that a slurry was prepared and this third slurry was used.
- TLC has been conventionally used as a main means for studying separation conditions by column chromatography and also for fractionation of target substances. Since the present invention can accurately detect the separation state of a target substance by a separation agent, which has been difficult to detect the separation state due to an optical response, the use of such a separation agent can be further expanded. It is expected to contribute to further development of separation and purification technology using such separation agents.
- Each state in an example of the spot detection method of the present invention RTB spot of the raffinate component of the tregar base ETB spot of the extract component of the tregar base RFRF spot of the raffinate component of flavanone EFLV spot of the raffinate component of flavanone STB racer base racemic Spot of Body SFLV Spot of Flavanone Racemate
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Abstract
Description
まず、ダイセル化学工業株式会社製CHIRALPAK IA(同社の登録商標)の充填剤(「IA充填剤」とも言う)4.00gと、石膏0.60gと、2%CMC(カルボキシメチルセルロース)1110(ダイセル化学工業株式会社製)水溶液4.00gと、20%スノーテックスC(日産化学工業株式会社製)水溶液0.60gとを、水0.40g、エタノール1.60gの混合溶液に添加し、超音波を照射しながら十分に攪拌して第一のスラリーを調製した。また、シリカゲル4.00g(ダイソー株式会社製液体クロマトグラフィー用、IR-60-5/20-U)、石膏0.20g、2%CMC(カルボキシメチルセルロース)1110(ダイセル化学工業株式会社製)水溶液6.00g、マンガン含有ケイ酸亜鉛0.04gとを、水2.02g、エタノール2.80gの混合溶液に添加し、超音波を照射しながら十分に攪拌して第二のスラリーを調製した。これらのスラリーを、仕切り板を装着したTLCプレート作製用スプレッダを用いてガラス板の表面に均一に塗布し、塗布したそれぞれのスラリー層を風乾し、真空ポンプで引きながら60℃で3時間真空乾燥することによって、第一のスラリーによる第一の分離剤層と、第二のスラリーによる、第一の分離剤層に併設された第二の分離剤層とを有するTLCプレート1を12枚形成した。
次に、第一のスラリーを、TLCプレート作製用スプレッダを用いてガラス板の表面に均一に塗布し、塗布したスラリー層を風乾し、真空ポンプで引きながら60℃で3時間真空乾燥し、幅が2cm、長さが10cm、厚さが150μmの、第一のスラリーによる第一の分離剤層のみを有するTLCプレート2を形成した。
さらに、IA充填剤に代えてダイセル化学工業株式会社製CHIRALPAK IC(同社の登録商標)の充填剤(「IC充填剤」とも言う)を用いた以外は第一のスラリーと同様にして第三のスラリーを調製し、この第三のスラリーを用いる以外はTLCプレート2と同様にして、第三のスラリーによる第三の分離剤層のみを有するTLCプレート3を形成した。そしてTLCプレート3を用いて、TLCプレート2による同時展開と同様に、トレガー塩基のラセミ体の1%の酢酸エチル溶液の約1μLとフラバノンのラセミ体の1%の酢酸エチル溶液とを同時に展開させ、シリカゲルシートへのスポットの転写を行った。なお、この充填剤とこの混合溶剤との組み合わせでは、カラムでもそれぞれの試料の光学異性体が分離しないことが判明している。
RTB トレガー塩基のラフィネート成分のスポット
ETB トレガー塩基のエクストラクト成分のスポット
RFLV フラバノンのラフィネート成分のスポット
EFLV フラバノンのラフィネート成分のスポット
STB トレガー塩基のラセミ体のスポット
SFLV フラバノンのラセミ体のスポット
Claims (8)
- 分離剤層を有するTLCプレートと、分離剤層に重ね合わせられて分離剤層のスポットが転写されるための被転写シートとを有し、
分離剤層は目的物質に対する分離性と紫外線又は発色試薬に対する光学応答性とを有し、
被転写シートは分離剤層とは異なる光学応答性と多孔性とを有する、スポット検出用セット。 - 被転写シートがシリカゲルシートであることを特徴とする請求項1に記載のスポット検出用セット。
- TLCプレートと被転写シートとを所定の位置関係で重ね合わせるための位置合わせ構造をさらに有することを特徴とする請求項1又は2に記載のスポット検出用セット。
- 請求項1~3のいずれか一項に記載のスポット検出用セットを用いて薄層クロマトグラフィーにおけるスポットを検出する方法であって、
TLCプレートの分離剤層に試料中の目的物質を展開させる工程と、
分離剤層の少なくとも目的物質を展開させた位置に被転写シートを重ね合わせる工程と、
重ね合わせられた分離剤層及び被転写シートを転写用溶剤で濡らす工程と、
分離剤層に重ね合わせられている被転写シート側から転写用溶剤を揮発させて分離剤層のスポットを被転写シートに転写する工程と、
被転写シートに転写されたスポットを光学的に検出する工程とを含む、スポット検出方法。 - 目的物質を展開させた分離剤層を、この分離剤層に被転写シートを重ね合わせる前に乾燥させる工程をさらに含むことを特徴とする請求項4に記載のスポット検出方法。
- TLCプレートに対する所定の位置関係で被転写シートを重ね合わせることを特徴とする請求項4又は5に記載のスポット検出方法。
- 目的物質に対する分離性と紫外線又は発色試薬に対する光学応答性とを有する分離剤層を備えたTLCプレートにおける分離剤層のスポットを分離剤層から転写するために用いるための、分離剤層とは異なる光学応答性と多孔性とを有する被転写シート。
- シリカゲルシートであることを特徴とする請求項7に記載の被転写シート。
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