DE112012002794T5 - Stain Detection Set, Stain Detection Method and Transfer Disc - Google Patents

Abstract

The position of a spot on a TLC plate is accurately detected by using a TLC plate having a separation media layer with specific optical response and further using a transfer disk with specific porosity that is placed on the separation media layer so that the stain on the separation media layer is transferred thereon, and by developing a target substance in a sample into the separation media layer of the TLC plate, the transfer disk is placed on the separation media layer at a position where at least the target substance has been developed, the applied separating media layer and the transfer disk are moistened with transfer solvent, the transfer solvent is evaporated from the side of the transfer disk placed on the separating media layer, to the stain on the separating media layer to transfer the transmission disc, and by the on the transfer The stain transferred to the disc is optically detected.

Description

Technical area

The present invention relates to a stain detection set for detecting a stain of a target substance in a release-agent layer without optical responsiveness in a TLC plate, a stain detection method using the same, and a transfer disc included in the set ,

Technical background

Thin layer chromatography (hereinafter also referred to as "TLC") is a method of separating and detecting specific components in mixtures. TLC is used to detect and isolate target substances in samples. In TLC, a spot of a target substance is detected by different optical responses between a release media layer of a TLC plate and the target substance. Thus, if the release media layer and the target substance have similar optical responses in TLC, it is not possible to detect the stain by optical response.

One attempt to solve this problem is a known TLC plate containing, on the same substrate, a first release layer having separability with respect to a target substance but no optical response, and a second release layer containing the release ability not having but having the optical responsiveness and formed adjacent thereto (see, for example, Patent Document 1). In this TLC plate, a target substance in a sample is developed from the first separation media layer toward the second separation media layer, and the spots separated in the first separation media layer migrate to and become dependent on the second separation media layer recorded optical response.

In this TLC plate, it is possible that an extracted component of samples easily absorbed by the first separation medium layer does not sufficiently reach the second separation medium layer. Furthermore, it is possible that the relative positions of patches in the first release media layer are not accurately maintained due to the differential migration speed of patches in the respective release media layers in the second release media layer. Accordingly, it is possible that this TLC plate does not allow accurate detection of the separation in the first release media layer, so that improvements can be made in these aspects.
Patent Document 1: Japanese Patent No. 3140138

Disclosure of the invention

The present invention provides a method that allows accurate detection of stains on TLC plates that do not permit detection of target substances by optical response.

the solution of the problem

The inventors of the present invention have come to the conclusion that the problem described above can be solved by transferring stains on the TLC plate to a transfer disc having optical responsiveness to a target substance, and thus have developed the present invention.

Thus, the present invention provides a stain detection kit including a TLC plate with a release media layer and a transfer disc placed on top of the release media layer so that a stain on the release media layer is transferred to it Separation media layer has separation properties with respect to a target substance and optical response to ultraviolet rays or a dye-forming reagent, and the transfer disc has optical response different from that of the separation media layer, and further has porosity.

The present invention further provides a stain detection kit wherein the transfer disc is a silica gel disc.

The present invention further provides the stain detection set further comprising an alignment mechanism for aligning the TLC plate and the transfer disc in a predetermined relative position.

The present invention further provides a stain detection method implemented in thin layer chromatography using the stain detection set of the present invention, the method comprising a step of developing a target substance in a sample into the release media layer of the TLC plate a step in which the transfer disk is placed on the release media layer at a position where at least the target substance has been developed, a step in which the applied release media layer and the transfer disk are moistened with a transfer solvent Step in which the transfer solvent comes from one side of the transfer disk applied to the release media layer is evaporated to transfer a spot on the release layer to the transfer disc, and includes a step in which the stain transferred to the transfer disc is optically detected.

The present invention further provides the stain detection method further including a step of drying the release-agent layer into which the target substance has been developed before the transfer-plate is placed on the release-medium layer.

The present invention further provides the patch detection method in which the transfer disc is placed on the TLC disc at a predetermined relative position.

The present invention further provides a transfer disk to which a mark is transferred on a release-agent layer of a TLC plate, this release-agent layer having release properties with respect to a target substance as well as optical response to ultraviolet rays or a chromogenic reagent, and the transfer disc optical response, which is different from that of the release media layer, as well as having porosity.

The present invention further provides the transfer disk, which is a silica gel disk.

In the present invention, the transfer wafer is applied to the release media layer of the TLC plate so that at least a portion of a stain on the release media layer is transferred to the transfer wafer by transferring the transfer solvent from the back of the transfer media Layer on the transfer disc is evaporated, and thus the spot on the TLC plate can be accurately detected, which otherwise does not allow detection of a target substance by means of optical response.

Brief description of the drawings

1 Fig. 13 is a view showing an example of the patch detection method of the present invention;

2 Fig. 12 is a view showing spots of Tröger's base on the TLC plate 1 having first and second separation media layers;

3 Fig. 12 is a view showing spots of flavanone on the TLC plate 1;

4 Fig. 13 is a view showing spots of Tröger's base and flavanone on the transfer disk to which stains have been transferred from the TLC plate 2; and

5 FIG. 14 is a view showing spots of Tröger's base and flavanone on the transfer disk to which stains have been transferred from the TLC plate 3. FIG.

Embodiment of the invention

The stain detection kit of the present invention includes a TLC plate having a release media layer and a transfer disc which is placed on the release media layer so that a stain on the release media layer is transferred thereon.

With respect to the TLC plate, there are no particular limitations as far as it is a TLC plate having a separation medium layer having release properties with respect to a target substance as well as optical response to ultraviolet rays or a chromogenic reagent. As used herein, the term "optical response to ultraviolet rays" refers to luminescence by ultraviolet rays, such as fluorescence, or absorption of ultraviolet rays. The term "optical response to a chromogenic reagent" as used herein refers to color development of the chromogenic reagent. The TLC plate may include a TLC plate having a substrate and a release media layer formed thereon. Accordingly, the TLC plate can be selected or prepared from known TLC plates by applying slurry of a separation medium having the releasability and the optical response to the substrate, and drying the slurry to form the separation medium layer.

The substrate may be a known substrate for TLC. The substrate may include, for example, a flat plate made of glass, plastics, metals or paper. There are no particular restrictions on the shape of the substrate, and it preferably has a rectangular shape which is commonly used for TLC.

The separation medium may be a particulate separation medium. The particulate separation medium may be in the form of particles consisting of a separation medium or particles comprising a separation medium held on a particulate support.

The separation medium can either be a low molecular weight or high molecular weight Separation medium having optical response. The low molecular weight separation medium may include, for example, ligand exchange separation media, charge transfer (π-π) separation media, hydrogen bonding separation media, clathrate separation media, ion-binding separation media, intercalation separation media, crown ethers or derivatives thereof, and cyclodextrins or derivatives thereof. The high molecular weight separation medium may include, for example, polysaccharide derivatives, polyamides, polymethacrylate esters, polyacrylamides, proteins, and tartaric acid derivatives.

The polysaccharide derivatives may include, for example, polysaccharide derivatives used as separation media of optical isomers and including a polysaccharide and aromatic ester, aromatic carbamoyl, aromatic ether and carbonyl groups, all or part of the hydroxyl or amino groups in the polysaccharide which may include, for example, phenyl carbonate 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 contain one or more substituents selected from the group consisting of hydrocarbons having 1 to 20 carbon atoms and halogens.

The support is preferably a porous material in order to improve the separation performance. The support may include, for example, synthetic polymers such as crosslinked polystyrenes, crosslinked acrylic polymers and epoxy polymers, polysaccharides such as cellulose and crosslinked and reinforced cellulose, crosslinked agarose, crosslinked dextran and crosslinked mannose (mannan), inorganic materials such as alumina, silica gel , mesoporous silica gel, diatomaceous earth, fused silica, clay mineral, zirconia and metals.

The particle diameter of the separation medium may be selected according to the purpose of separation on the TLC plate. For example, to study the conditions for column chromatography at moderate pressures, the particle diameter is preferably 10 μm or more, more preferably 10 to 100 μm, and more preferably 20 to 100 μm, to obtain results that can be used for chromatography at moderate pressures. The particle diameter of the separation medium may be an average particle diameter or a catalog value measured on conventional particle diameter analyzers.

In preparing the release media layer, it can be prepared by a known method of fabricating TLC plates, for example, by applying slurry containing the release medium and an application solvent to the surface of a support using a coater, or by applying the slurry is sprayed onto the surface of the substrate.

The application solvent may be water, an organic solvent or a mixed solvent thereof. The organic solvent may include, for example, 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, esters such as ethyl acetate, amides such as dimethylformamide, hydrocarbons such as pentane, hexane and petroleum ether, aromatic hydrocarbons such as benzene, toluene and xylene, and halogen-containing compounds such as methylene chloride, chloroform, bromoform, chlorobenzene and bromobenzene.

The applying solvent is preferably a solvent mixed with water and a water-soluble organic solvent, preferably a solvent mixed with water and an alcohol, and more preferably with a solvent mixed with water and ethanol. The content of the alcohol in the mixed solvent is preferably 0.1 to 50% by weight, more preferably 10 to 40% by weight, and still more preferably 20 to 30% by weight.

When two release media layers are formed contiguously on a substrate, the slurries for forming these release media layers preferably contain the same type of deposition solvent and preferably contain the deposition solvent of the same composition to minimize the disruption at the interface of the separation media. To prevent layers and thus to avoid any influence on the migration of stains in the release media layers.

The content of the application solvent in the slurry may be determined according to the uniformity of the formed release layer, the thickness of the layer, and economical aspects, and is preferably 10 to 5,000 parts by weight, preferably 50 to 1,000 parts by weight, and more preferably 100 to 300 parts by weight relative to 100 parts by weight of the separation medium.

Preferably, the slurry further contains a binder to improve the strength of the formed release media layer. The binder may be a component that can impart binding capacity to the layer of the Form separation medium on the surface of the substrate. The binder may include organic binders such as gypsum and colloidal silica, organic binders such as organic fibers, e.g. Cellulosic microfibrils, thickeners, e.g. Alkaline water-soluble copolymers, hydroxyethyl cellulose and carboxymethyl cellulose, polyvinyl alcohols and acrylic acids. One or more types of binder may be used.

The content of the binder in the slurry may be selected according to the type of the binder from the viewpoint of the strength of the formed release media layer and a suitable rate of ascent of the mobile phase in the release media layer. For example, when gypsum is used, the content of the binder is preferably 0.1 to 50 parts by weight, more preferably 5 to 30 parts by weight, and more preferably 10 to 20 parts by weight relative to 100 parts by weight of the separation medium. When an organic binder such as carboxymethyl cellulose is used, the content of the binder is preferably 0.1 to 50 parts by weight, more preferably 0.5 to 10 parts by weight, and more preferably 1 to 3 parts by weight relative to 100 parts by weight of the separation medium.

The transfer disk has optical response different from that of the release media layer and porosity. The term "optical response different from" means that an optical reaction resulting from irradiation with ultraviolet rays or color developing treatment with a homogeneous reagent is different from the other optical reaction so as to be colored or colored Brightness can be distinguished from each other.

The "porosity" of the transfer disc expresses that, when the transfer disc is placed on the release media layer and then moistened with a solvent and dried from the side of the transfer disc, the transfer disc has spaces therebetween, so that a solvent of the Transfer disc is absorbed at an approximate speed at which at least a portion of the component (s) forming a stain on the release media layer is absorbed by the transfer disc at a speed that allows transfer of the component (s) to the transfer disc. In view of this, the overall porosity of the transfer wheel is preferably 0.1 to 0.9, more preferably 0.2 to 0.8, and still more preferably 0.4 to 0.8. The total porosity of the transfer disc can be calculated, for example, from the weight difference between the weight of the transfer disc that has absorbed the solvent and the weight of the transfer disc, the specific gravity of the absorbed solvent, and the volume of the transfer disc calculated from the outer dimensions thereof.

In view of the migration of components which form stains from the TLC plate, the porosity of the transfer disc is preferably uniform at the portion resting on the TLC plate and is preferably uniform throughout the transfer disc (for example, the transfer disc has a uniform overall porosity ).

The transfer disc can be manufactured by various methods. For example, the transfer wheel may be made by applying slurry of the above-described support, such as silica gel, to a porous support and drying the slurry. The porous substrate may include, for example, pore-punched plates, sintered plates, meshes (meshes), and porous films. The material of the substrate may include, for example, glass, plastics, metals and ceramics.

The transfer wheel may also be made by applying slurry containing the support, the application solvent and optionally the binder or an aggregate to a substrate and stripping the post-drying layer from the substrate. The aggregate may contain, for example, meshes and fillers (fibers). The content of the binder in the slurry for the transfer sheet is preferably 0.1 to 50 parts by weight, more preferably 0.5 to 30 parts by weight, and more preferably 1 to 20 parts by weight relative to 100 parts by weight of the support from the viewpoint of the strength of the transfer sheet. The content of the aggregate in the slurry for the transfer sheet is preferably 0.1 to 0.9 parts by weight, more preferably 0.2 to 0.8 parts by weight, and more preferably 0.3 to 0.7 from the viewpoint of the strength of the transfer sheet Parts by weight relative to 100 parts by weight of the carrier.

The transfer disc can also be made as a porous silica gel (monolithic plate) which is formed into a disc shape by the sol-gel method.

Of these transfer disks, the transfer disk is preferably a silica gel disk, which is a silica gel in disk form, formed by any of the above is made to adapt the overall porosity of the transfer disc and the affinity for a solvent.

The transfer sheet preferably has a thickness of 10 to 2,000 μm, more preferably 50 to 1,000 μm, and more preferably 100 to 500 μm, in terms of permeability to the target substance, prevention of scattering of the spot of the target substance, and strength of the transfer disc.

When the transfer disk slurry is manufactured for the transfer disk, the carrier preferably has a particle diameter of 0.1 to 100 μm, more preferably 1 to 50 μm, and still in terms of porosity and proper adhesiveness between the transfer disk and the TLC plate better 5 to 40 microns.

The transfer wafer further preferably includes an indicator that fluoresces when a spot is detected by optical responsivity upon exposure to ultraviolet rays to enable more accurate detection. The fluorescent indicator may be a known fluorescent indicator, which may include, for example, magnesium tungstate, manganese-containing zinc silicate, and the like. The transfer disc can be made by further adding the fluorescent indicator to the transfer disc slurry. The content of the fluorescent indicator can be selected within the range that allows separation of the target substance, and is generally 0.1 to 5 parts by weight relative to 100 parts by weight of the carrier.

When a stain is detected by optical response due to the use of a chromogenic reagent, known methods of chromogenic reagents and color development treatment can be applied to the transfer disc. The chromogenic reagent may include, for example, anisaldehyde solution, molybdophosphoric acid solution, iodine, ninhydrin solution, chameleon solution, DNPH solution, manganese chloride solution and bromocresol green solution. Color development treatment may include, for example, treatment, including application of the chromogenic reagent to the transfer wheel by application, spraying or exposure, and optional heating of the transfer wheel, which permits color development.

Preferably, the stain detection set of the present invention further includes an alignment mechanism for aligning the TLC plate and the transfer disc to more accurately detect the stain in the release media layer at a predetermined relative position. For example, the alignment mechanism may include a mechanism for determining the position where the TLC plate overlaps the transfer disk and shifting it in the horizontal direction. That is, the alignment mechanism may include a mechanism by which the TLC plate is attached to the transfer disc at ends thereof by means of a hinge, a frame holding the TLC plate and the overlying transfer disc in the horizontal direction, a protruding portion provided on either the TLC plate or the transfer disc, such as a bead, and a recessed portion provided on the other member, such as a hole, and an adhesive means releasably adhering the TLC Plate and the transfer disc allows, such as an adhesive tape or a Velcro.

The stain detection method of the present invention can be carried out with the above-described stain detection set of the present invention. The stain detection method of the present invention comprises the first step, in which a target substance in a sample is developed into the release-agent layer of the TLC plate, the second step, in which the transfer disc is placed on the release-agent layer at a position on the release plate The third step, in which the release-agent layer and the transfer disc, which are lying on top of each other, are moistened with a transfer solvent, the fourth step, in which the transference layer is deposited on which at least the target substance has been developed. Solvent is volatilized from the side of the transfer disk resting on the release media layer to transfer a stain on the transfer medium layer to the transfer disk, and the fifth step, in which the stain transferred to the transfer disk is optically detected.

The first step can be carried out in a similar manner to conventional thin-layer chromatography. That is, the first step may be carried out by applying a spot of a sample to an edge of the release media layer, optionally drying the spot of the sample, impregnating the release media layer in a mobile phase with the edge on which the Stain the sample has been applied, and the target substance in the sample is developed into the release media layer inside. The target substance is a substance that is visually appealing in the release media layer in the same manner as the release media layer.

In the second step, it is sufficient to place the transfer disc in a position at which the target substance has been developed into the release media layer. The transfer disc may be placed on a portion of the release-agent layer, such as a portion where the mobile phase has been developed, but is preferably applied to the entire release-agent layer to detect the position of a stain. The second step is preferably a step in which the transfer disc is placed at a predetermined position relative to the TLC plate to allow more accurate detection of the position of a spot in the release media layer.

The third step may be carried out by spraying the transfer solvent onto the entire transfer disk from the side of the transfer disk deposited on the release media layer, or by soaking the transfer disk and the release media layer in the transfer solvent, or by the transfer disc is moistened with the transfer solvent prior to placement, so that the release media layer is sufficiently moist after being placed on the transfer medium.

The transfer solvent may be a solvent that allows elution of at least some of the components that form a stain in the release media layer from the release media layer. The transfer solvent may be the above-described mobile phase or a known organic solvent or a mixture of two or more members selected from the group consisting of known solvents, water, acids and bases. The transfer solvent may be an alcohol such as ethanol, methanol and 2-propanol, THF, ethyl acetate, chloroform or acetone in view of the solubility of the target substance and the elution ability of the target substance. When the release-agent layer is made to be a coated material consisting of the support which accommodates the polysaccharide-derivative separation medium, the transferring solvent is preferably an alcohol such that the polysaccharide derivative-containing material Separation medium is not dissolved on the support.

The fourth step is preferably carried out by air-drying the transfer disk resting on the release media layer from the side of the transfer disk to achieve absorption of the transfer solvent by the transfer disk at a corresponding speed, with air drying to improve the transferred amount with cool air of 10 to 30 ° C is preferred. Air drying may be performed on the entire transferred plate or on a portion of the transferred plate where the stain may be present.

The fifth step may be carried out while the transfer disk is resting on the release media disk or after the transfer disk has been removed. In the fifth step, the target substance has optical response that is detectably different from that of the transfer disc. The optical detection in the fifth step, when the contact plane of the transfer disc with the separation medium plate is the upper side, may be performed on the upper side or the back side of the transfer disc in the area where optical response of the transferred spot is possible.

The stain detection method of the present invention may include other steps as far as the effect of the present invention can be achieved. Other steps may include the step of drying the release media layer to which the target substance has been applied before the transfer disc is placed on the release media layer. This drying step is preferably integrated to allow more accurate detection of the location of the stain in the release media layer. The step of drying is preferably the step of drying with warm air of from 20 to 80 ° C to improve the operation, or preferably, the step of drying with cold air in the aspect of safety.

The patch detection method of the present invention may be carried out by, for example, as shown in FIG 1 to produce a TLC plate containing a separation gel layer of silica gel to which a polysaccharide derivative is applied (A in 1 ), a spot of a sample, such as a solution of racemic optical isomers, is applied to one edge of the release media layer in the TLC plate, the optical isomers in the sample are developed from the edge to the other edge, and with cooler Air is dried by a dryer (B in 1 ), a transfer disk such as a silica gel disk is placed on the release media layer in the TLC disk (C in FIG 1 ), a transfer solvent such as ethanol is sprayed on the entire back surface of the overlying transfer disk to wet both the transfer disk and the release media layer with the transfer solvent (D in FIG 1 ), the transfer disk and the release media layer, which are superposed, are gradually dried from the back of the transfer disk with cool air from a dryer (E in 1 ), and by irradiating the rear side of the transfer disk with UV light, to optically detect a stain transferred to the transfer disk (F in 1 ).

The optical detection of the spot on the transfer disk in the present invention can be used to detect the separation result on the TLC disk. According to the present invention, both spots of a raffinate component and an extract component, if they are on the TLC plate, including the positional relationship thereof resulting from separation by TLC, can be detected accurately. Further, according to the present invention, the respective separation of a plurality of samples can be accurately detected when stains of the sample are applied in series to the TLC plate and developed simultaneously. The target substance may also be isolated by detecting the part including a certain spot transferred to the transfer plate and performing extraction operations. Furthermore, the target substance may also be isolated by detecting the part of the release-agent layer corresponding to the part containing the particular spot detected on the transfer-plate.

Examples

Testing the R _F value

First, 4.00 g of the filler were (hereinafter also referred to as "IA-filler") CHIRALPAK IA ^® (Daicel Corporation) 0.60 g of gypsum, 4.00 g of a 2% aqueous solution of CMC (carboxymethyl cellulose 1110 (Daicel Corporation) and 0.60 g of a 20% aqueous solution of Snowtex C (Nissan Chemical Industries, Ltd.) was added to a mixed solution of 0.40 g of water and 1.60 g of ethanol and thoroughly stirred while simultaneously Ultrasound acted to produce a first slurry. Furthermore, 4.00 g of silica gel (for liquid chromatography, from Daiso Co., Ltd., IR-60-5 / 20-U), 0.20 g of gypsum, 6.00 g of a 2% aqueous solution of CMC ( Carboxymethyl cellulose 1110 (Daicel Corporation) and 0.04 g manganese-containing zinc silicate were added to a mixed solution of 2.02 g water and 2.80 g ethanol and stirred thoroughly while sonication to produce a second slurry. These slurries were uniformly coated on a partitioned glass plate using a TLC plate-making applicator, the dispersed layers of the slurries were air dried at 60 ° C for 3 hours, and vacuum-dried while being vacuumed by 12 TLC with a vacuum pump Plates 1, each containing a first separation media layer of the first slurry and a second separation media layer of the second slurry, which was present adjacent to the first release media layer.

The first release media layer has a width of 2 cm, a length of 10 cm and a thickness of 150 μm, and the second release media layer has a width of 3 cm, a length of 10 cm and a thickness of 110 μm. The first release media layer corresponds to the layer of IA filler, and the second release media layer corresponds to the silica gel layer. The IA filler has an average particle diameter of 20 μm, and the silica gel has an average particle diameter of 14.4 μm.

A 1% solution of Tröger's base (TB) racemic substance in ethyl acetate (approximately 1 μl) was spotted onto TLC plate 1 at approximately 1.0 cm from the lower end in the longitudinal direction. The TLC plate 1 was set in a developing vessel containing a mixed solvent containing n-hexane and ethanol in a volume ratio of 9: 1 with the sample spot at the bottom, so that optical isomers of the Tröger's base in the sample in the longitudinal direction of the first release media layer have been developed. After the first development, the TLC plate 1 was once dried with cool air. The TLC plate was then placed in a development vessel containing ethanol with the first separation media layer down so that the Tröger base optical isomers were further developed from the first separation media layer to the second separation media layer.

After the second development, the TLC plate 1 was dried with cool air and irradiated with ultraviolet rays. As a result, the stains of the raffinate component RTB and the extract component ETB, which had migrated from the first separation layer to the second separation layer, were perceived as green stains in the second release layer (FIG. 2 ). The first release layer absorbed ultraviolet rays and thus appeared as a black area. The R _F value of the stains was determined from the position at which the sample was spotted on the first release layer, the position to which the developing solution reached, and the center positions of the stains on the second release layer; as a result, the raffinate component and the extract component of Tröger's base had an R _F value of about 0.5 and about 0.35, respectively.

A 1% solution of racemic flavanone (FLV) in ethyl acetate (about 1 μl) was spotted in spots in the same manner as described above on another TLC plate 1 which became the racemic substance of flavanone in the second release media layer, and the positions of the raffinate component RFLV and the extract component EFLV in the second release media layer were examined. As a result, both components were perceived as green spots on the second release layer ( 3 ). The raffinate component and flavanone extract component had an R _F value of about 0.3 and about 1.75, respectively.

example 1

Then, the first slurry was uniformly applied to the surface of a glass plate by using a TLC plate-making coater, the coated layer of the slurry was air dried at 60 ° C for 3 hours, and vacuum-dried while vacuuming with a vacuum pump to obtain a TLC To prepare sheet 2 containing only the first release-media layer of the first slurry and having a width of 2 cm, a length of 10 cm and a thickness of 150 μm.

A 1% solution of racemic substance from Tröger's base in ethyl acetate (about 1 μl) and a 1% solution of racemic from flavanone in ethyl acetate (about 1 μl) were each at about 1.0 cm from the bottom in the longitudinal direction applied in spots on the TLC plate 2. The TLC plate 2 was set in a developing vessel containing a mixed solvent containing n-hexane and ethanol in a volume ratio of 9: 1 with the spots of the samples at the bottom so that both samples simultaneously in the longitudinal direction of the TLC plate 2 were developed. After development, the TLC plate 2 was dried with cool air to complete the development of stains.

On the first release media layer of this TLC plate 2 was placed a silica gel disc of the same size in a rectangular shape, ethanol was sprayed on the entire laid silica gel disc to both the silica gel disc and the first separation media layer Cool air was directed to the silica gel disk while resting on the first release media layer to dry both the first release media layer and the silica gel disk, and thus the stains on the first release media layer transferred from the first release media layer on the silica gel disc.

The silica gel disc was prepared by applying the second slurry to a glass plate in a similar manner as described above, drying the thus-formed second separation media layer having a thickness of about 300 μm and peeling it from the glass plate to adjust the layer in that it had a rectangular shape.

When the ethanol had dried, the silica gel disc was released from the TLC plate 2, and the contact plane of the silica gel disc with the TLC plate 2 was illuminated with ultraviolet rays. For example, raffinate components RTB and RFLV and extract components ETB and EFLV from Trögerscher base and flavanone were detected as green spots ( 4 ).

reference example

A third slurry was prepared in the same manner as in the preparation of the first slurry, except that the filling material (hereinafter also referred to as "IC filler") CHIRALPAK IC ^® (Daicel Corporation) was used instead of the IA-fill material. A TLC plate 3 was fabricated in the same manner as in the fabrication of the TLC plate 2 except for using the third slurry containing only a third release layer of the third slurry. A 1% solution of racemic substance from Tröger's base in ethyl acetate (approximately 1 μl) and a 1% solution of racemic from flavanone in ethyl acetate were grown in the same manner as in the co-development on TLC plate 2 simultaneously on the TLC plate 3, and the spots were transferred to the silica gel disk. It has been known that with the above-described combination of the filler material and the mixed solvent separation of the optical isomers in these samples is not possible even when using columns.

When the ethanol was dried, the silica gel disc was detached from the TLC plate 3, and the contact plane of the silica gel disc with the TLC plate 3 was illuminated with ultraviolet rays. Thus, Tröger's base or flavanone were detected without dissolution as one green spot STB or SFLV ( 5 ).

Applications in the industry

TLC has conventionally been used as a means of examining separation conditions in column chromatography as well as isolating target substances. The present invention enables more accurate detection of the separation of target substances than in prior art methods when employing separation media in which the detection of separation by optical reactions has heretofore been difficult. Therefore, it is expected that the present invention will contribute to the further dissemination of the use of such release media and to the further development of separation and purification processes using such release media.

Explanation of the reference numbers

• A to F

  each state during an exemplary stain detection method of the present invention

  RTB

  Stain of the raffinate component from Tröger's base

  ETB

  Stain of the extract component from Tröger's base

  RFLV

  Blot of the raffinate component from Flavanon

  EFLV

  Blot of the raffinate component from Flavanon

  STB

  Stain of racemic substance from Tröger's base

  SFLV

  Patch of racemic substance from flavanone

Claims (8)

A stain detection kit comprising a TLC plate with a release media layer and a transfer disc placed on the release media layer such that a stain on the release media layer is transferred thereon, wherein the release media layer has separation properties with respect to a target substance and optical response to ultraviolet rays or chromogenic reagent, and the transfer disk has optical response different from that of the release media layer, and further has porosity. A stain detection kit according to claim 1, wherein the transfer disc is a silica gel disc. A stain detection set according to claim 1 or 2, further comprising an alignment mechanism for aligning the TLC plate and the transfer disc at a predetermined relative position. A stain detection method implemented in thin layer chromatography using the stain detection set of any one of claims 1 to 3, the method comprising: a step of developing a target substance in a sample into the release media layer of the TLC plate; a step of placing the transfer disk on the release media layer at a position where at least the target substance has been developed; a step of wetting the applied release media layer and the transfer disk with a transfer solvent; a step in which the transfer solvent is evaporated from one side of the transfer disc placed on the separation media layer to transfer a spot on the release media layer to the transfer disc; and a step of optically detecting the stain transferred to the transfer disk. The spot detection method according to claim 4, further comprising a step of drying the release-agent layer into which the target substance has been developed before the transfer-plate is placed on the release-agent layer. The spot detection method according to claim 4 or 5, wherein the transfer disc is placed on the TLC plate at a predetermined relative position. A transfer wafer to which a spot is transferred on a release media layer of a TLC plate, said release media layer having separation properties with respect to a target substance and further having optical response to ultraviolet rays or a homogeneous reagent, and the transfer disc having optical response; which is different from that of the release media layer and has porosity. Transfer disc according to claim 7, which is a silica gel disc.

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