CN103649746B - Spot detection external member, spot detection method and transfer sheet - Google Patents
Spot detection external member, spot detection method and transfer sheet Download PDFInfo
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- CN103649746B CN103649746B CN201280032952.0A CN201280032952A CN103649746B CN 103649746 B CN103649746 B CN 103649746B CN 201280032952 A CN201280032952 A CN 201280032952A CN 103649746 B CN103649746 B CN 103649746B
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Abstract
Accurately detect by the following method the position of the spot on TLC plate: use the TLC plate with separating medium layer (it has particular optical response), and also use the transfer sheet (this transfer sheet will cover on this separating medium layer so that the spot on separating medium layer is transferred on it) with specific pore rate, and the target substance in sample is expanded in the separating medium layer of TLC plate; In the position that at least this target substance has launched, this transfer sheet is covered on separating medium layer; With shifting capped separating medium layer and the transfer sheet of wet with solvent; Make this transfer solvent evaporates so that the spot on separating medium layer is transferred to transfer sheet from transfer sheet one side covering separating medium layer; And optical detection is transferred to the spot of transfer sheet.
Description
Technical field
The present invention relates to for detection of the spot in TLC plate without target substance in the separating medium layer of optic responsePoint spot detection external member, use its spot detection method and be included in the transfer sheet in this external member.
Background technology
Thin-layered chromatography (hereinafter also referred to as " TLC ") is the known method of specific components in separation and detection mixture.TLC for detection of with sample separation in target substance. In TLC, between separating medium layer and target substance by TLC plateDifferent optic responses detects the spot of target substance. Thus, when separating medium layer and target substance have similar in TLCOptic response time, this spot can not detect by optic response.
In order to solve problems, on same substrate, contain to adjoin and formed thereon target substance is had to separationCharacter but do not there is the first separating medium layer of optic response and do not there is separating property but there is second of optic responseThe TLC plate of separating medium layer is known (referring to for example patent documentation 1). In this TLC plate, the target substance in sample is fromOne separating medium layer is deployed into the second separating medium layer, and detects and move to the second separating medium layer according to its optic responseIn the spot separating in the first separating medium layer.
In this TLC plate, in sample, easily may not can be arrived fully by the extraction components of the first separating medium layer absorptionReach the second separating medium layer. In addition, due to the different migration velocities of spot in each separating medium layer, spot in the first separating medium layerThe relative position of point may not can accurately remains in the second separating medium layer. Therefore, this TLC plate may not can be examined exactlySurvey the separation in the first separating medium layer, left in these areas improved space.
Patent documentation 1: Japanese Patent No. 3140138.
Summary of the invention
The invention provides and can accurately detect and can not detect spot on the TLC plate of target substance by optic responseA kind of technology.
The method of technical solution problem
The inventor has been found that can be by transferring to the spot on TLC plate target substance is had to optic responseIn the transfer sheet of property, address the above problem, complete thus the present invention.
Thus, the invention provides spot detection external member, comprise and there is the TLC plate of separating medium layer and will cover described pointFrom on dielectric layer so that the spot on described separating medium layer is transferred to the transfer sheet on it, wherein said separating medium layer hasFor the separating property of target substance and the optic response to ultraviolet ray or colour reagent, and described transfer sheet has differenceOptic response in described separating medium layer also also has porous.
The present invention also provides the spot detection external member that wherein this transfer sheet is silica gel piece.
The present invention also provide further comprise for by described TLC plate and described transfer sheet at predetermined relative position place pairThe spot detection external member of accurate aligning guide.
The present invention also provides the spot detection external member of the application of the invention and the spot inspection implemented in thin-layered chromatographySurvey method, the method comprises: the target substance in sample is expanded to the step in the described separating medium layer of described TLC plate;Described transfer sheet is covered the step on described separating medium layer by the position of having launched at least described target substance; WithShift the capped separating medium layer of wet with solvent and the step of transfer sheet; From cover described separating medium layer described in turnMoving sheet one side makes described transfer solvent evaporates the spot on described separating medium layer is transferred to the step of described transfer sheet; LightLearn the step that detects the spot that is transferred to described transfer sheet.
The present invention also provides spot detection method, is further included in described transfer sheet is covered to described separating medium layerThe step of dry described separating medium layer before upper, wherein, target substance has been expanded in described separating medium layer.
The present invention also provides spot detection method, and wherein said transfer sheet is described in predetermined relative position place coversOn TLC plate.
The present invention also provides and the spot on the separating medium layer of TLC plate is transferred to the transfer sheet on it, this separating mediumLayer has for the separating property of target substance and the optic response to ultraviolet ray or colour reagent, and this transfer sheet hasBe different from optic response and the porous of described separating medium layer.
The present invention also provides transfer sheet, and it is silica gel piece.
The present invention uses the transfer sheet covering on the separating medium layer of TLC plate, makes by from covering separating mediumThe back side of the transfer sheet on layer this transfer solvent that volatilizees, turns thereby at least a portion spot on separating medium layer is transferred to thisMove on sheet, and can accurately detect thus and can not detect the spot on this TLC plate of target substance by means of optic responsePoint.
Brief description of the drawings
Fig. 1 is the view that shows an example of spot detection method of the present invention;
Fig. 2 is the view that is presented at the spot of the Troeger alkali on the TLC plate 1 with the first and second separating medium layers;
Fig. 3 is the view that is presented at the spot of the flavanones on TLC plate 1;
Fig. 4 is presented at spot to transfer to Troeger alkali in the transfer sheet it and flavanones from TLC plate 2The view of spot; With
Fig. 5 is presented at spot to transfer to Troeger alkali in the transfer sheet it and flavanones from TLC plate 3The view of spot.
Detailed description of the invention
Spot detection external member of the present invention comprises: have the TLC plate of separating medium layer and cover on this separating medium layerSo that the spot on separating medium layer is transferred to the transfer sheet on it.
This TLC plate is not particularly limited, as long as it is that TLC plate and this separating medium layer with separating medium layer hasFor the separating property of target substance and the optic response to ultraviolet ray or colour reagent. Term used is " to ultraviolet hereinThe optic response of line " refer to by luminescence-utraviolet (as fluorescence) or absorb ultraviolet ray. Term used is " to colour developing examination hereinThe optic response of agent " refer to the colour developing of colour reagent. This TLC plate can comprise having substrate and Jie that separates who forms thereonThe TLC plate of matter layer. This TLC plate can suitably be selected in known TLC plate, or has this separation by applying on substrateThe slurry of the separating medium of character and optic response dry this slurry obtain to form this separating medium layer.
This substrate can be the known substrate for TLC. This substrate can comprise for example by glass, resin, metal or paperOpen the flat board of making. The shape of substrate is not particularly limited, and is preferably generally used for the rectangular shape of TLC.
This separating medium can be graininess separating medium. This graininess separating medium can be made up of separating mediumParticle, comprise the particle that is fixed on the separating medium on pelleted substrate.
This separating medium can be low-molecular-weight or the HMW separating medium with optic response. Low-molecular-weight is dividedFrom medium for example can comprise ligand exchange type separating medium, electric charge shift (π-π) type separating medium, hydrogen bond type separating medium,Inclusion compound type separating medium, ion of bonding separating medium, insert type separating medium, crown ether or derivatives thereof and cyclodextrin or itsDerivative. HMW separating medium can comprise for example polysaccharide derivates, polyamide, polymethacrylates, polyacrylamideAmine, protein and tartaric acid derivatives.
This polysaccharide derivates can comprise, for example, is used as the separating medium of optical isomer and comprises polysaccharide and be somebody's turn to do with replacementAny in polysaccharide in the aromatic ester of all or part hydroxyl or amino group, aromatic carbomoyl-, aromatic oxide and carbonylPolysaccharide derivates, it can comprise for example cellulosic carbanilate derivative, cellulosic phenyl ester derivatives, straightThe carbanilate derivative of chain starch and the phenyl ester derivatives of amylose. Phenyl in these derivatives can containThere is one or more to be selected from the substituting group in hydrocarbon and the halogen with 1 to 20 carbon atom.
Consider and improve separating property, preferably porous material of this carrier. This carrier can comprise for example synthetic macromolecule,As crosslinked polystyrene, crosslinked acrylic acid polymer and epoxy polymer; Polysaccharide, as cellulose and the fiber that is cross-linked and strengthensElement, Sepharose, cross-link dextran and crosslinked mannosan; Inorganic material, as aluminium oxide, silica gel, mesoporous silica gel(mesoporoussilicagel), zeolite, diatomite, fused silica, clay mineral, zirconia and metal.
Can select according to the separation object on TLC plate the particle diameter of this separating medium. For example press in order to study in appropriatenessThe condition of the column chromatography under power, considers and obtains the chromatographic result being applicable under modest pressure, this particle diameter is preferably 10μ m or larger, more preferably 10 to 100 μ m, more more preferably 20 to 100 μ m. The particle diameter of this separating medium can be at conventional particle diameterThe average grain diameter recording on analyzer, or can be catalogue value.
The in the situation that of this separating medium layer of preparation, it can, by known TLC plate preparation method preparation, for example, lead toCross and on supporting mass surface, apply the slurry that contains this separating medium and apply solvent with spreader, or by this slurry is sprayedTo supporting mass surface.
This applies solvent can be water, organic solvent and mixed solvent thereof. This organic solvent can comprise for example alcohol, as firstAlcohol, ethanol and isopropyl alcohol; Ketone, as acetone and MEK; Ether, as oxolane and dioxane; Nitrile, as acetonitrile; Sulfoxide,As methyl-sulfoxide; Sulfone, as sulfolane; Ester, as ethyl acetate; Acid amides, as dimethyl formamide; Hydrocarbon, as pentane, hexane and oilEther; Aromatic hydrocarbon, as benzene, toluene and dimethylbenzene; Halogen contained compound, as carrene, chloroform, bromofom, chlorobenzene and bromobenzene.
This applies the preferably mixed solvent of water and water-miscible organic solvent of solvent, is more preferably water molten with mixing of alcoholAgent, then be more preferably the mixed solvent of water and ethanol. In this mixed solvent, the content of alcohol is preferably 0.1 to 50 quality %, more preferably10 to 40 quality %, more more preferably 20 to 30 quality %.
When adjoining while forming two separating medium layers, consider the disorder that prevents separating medium layer boundary on substrateAnd preventing thus any impact on spot migration in separating medium layer, the slurry that is used to form these separating medium layers preferably containsThere is the solvent that applies of same type, more preferably contain the solvent that applies with same composition.
The content that applies solvent in slurry can be according to thickness and the economical view-point of the uniformity of the separating medium layer forming, layerPut and determine, and be preferably 10 to 5000 mass parts with respect to the separating medium of 100 mass parts, more preferably 50 to 1000 qualityPart, more more preferably 100 to 300 mass parts.
Consider the intensity of improving the separating medium layer that forms, preferably this slurry further contains adhesive. This is stickyMixture can be to provide bond property to form the component of separating medium layer on substrate surface. This adhesive can comprise nothingMachine adhesive, as gypsum and colloidal silica; Organic bond, as organic fiber, for example fibrillation cellulose; Thickener,For example alkaline water soluble copolymer, hydroxyethylcellulose and carboxymethyl cellulose; Polyvinyl alcohol and acrylic acid. Can use oneOr the adhesive of more kinds of types.
Consider the suitable rate of climb of mobile phase in the intensity of formed separating medium layer and separating medium layer, in slurryBinder content can suitably select according to the type of adhesive. For example, in the time using gypsum, the content of adhesive is relativeBe preferably 0.1 to 50 mass parts, more preferably 5 to 30 mass parts and more preferably 10 to 20 matter again in the separating medium of 100 mass partsAmount part. In the time using organic bond as carboxymethyl cellulose, the content of adhesive is excellent with respect to the separating medium of 100 mass partsElect 0.1 to 50 mass parts, more preferably 0.5 to 10 mass parts and more preferably 1 to 3 mass parts again as.
This transfer sheet has the optic response that is different from separating medium layer, and has porous. Phrase " be different from ...Optic response " refer to by the optic response that ultraviolet ray is irradiated or the color development treatment of colour reagent causes and be different from other lightLearn response, to be optically distinguished from each other by means of color or brightness.
" porous " of transfer sheet means when this transfer sheet covers subsequently to be done by wet with solvent and from transfer sheet one sideOn dry separating medium layer time, this transfer sheet has space, absorbs, in this speed to make solvent be transferred sheet with suitable speedUnder degree, form at least a portion of the component of spot on separating medium layer to allow this component to transfer to the speed quilt in transfer sheetThis transfer sheet absorbs. Consider above-mentioned factor, the overall porosity of this transfer sheet be preferably 0.1 to 0.9, more preferably 0.2 to 0.8 andAgain more preferably 0.4 to 0.8. The overall porosity of transfer sheet can be by weight and the transfer sheet weight of transfer sheet that has for example absorbed solventThe proportion of the weight difference between amount, the solvent absorbing and the transfer sheet volume being calculated by its outside dimension calculate.
Consider the component that forms spot from the migration of TLC plate, the porosity of this transfer sheet is preferably covering on TLC platePart place is uniform (for example, this transfer sheet has uniform overall porosity), and on whole transfer blade, is more preferably even.
This transfer sheet can be prepared by the whole bag of tricks. For example, this transfer sheet can be passed through above-mentioned carrier (as silica gel)Slurry be applied on porous supporting mass and prepared by dry this slurry. This porous supporting mass can comprise for example by boringBe provided with plate, sintered plate, net (net) and the perforated membrane of hole. The material of this supporting mass for example can comprise glass, plastics,Metal and pottery.
This transfer sheet can also by by contain described carrier, described in apply solvent and optional adhesive or the slurry of aggregateMaterial is applied on substrate and peels off from substrate the layer obtaining after dry and prepare. This aggregate can comprise for example net and fill outMaterial (fiber). Consider the intensity of transfer sheet, the institute for the content of the slurry adhesive of transfer sheet with respect to 100 mass partsState carrier and be preferably 0.1 to 50 mass parts, more preferably 0.5 to 30 mass parts and more preferably 1 to 20 mass parts again. Consider transferThe intensity of sheet, is preferably 0.1 to 0.9 for the content of the slurry aggregate of transfer sheet with respect to the described carrier of 100 mass partsMass parts, more preferably 0.2 to 0.8 mass parts and more preferably 0.3 to 0.7 mass parts again.
This transfer sheet can also be shaped to by sol-gal process sheet Bio-sil (entire plate) form obtain.
In these transfer sheet, to consider and regulate the overall porosity of transfer sheet and the affinity with solvent, this transfer sheet is excellentChoosing is the silica gel piece as the sheet silica gel obtaining by any said method.
Consider target substance permeability, prevent the intensity of target substance spot diffusion and transfer sheet, this transfer sheet is excellentChoosing has 10 to 2000 μ m, more preferably 50 to 1000 μ m and the more preferably thickness of 100 to 500 μ m again.
In the time preparing this transfer sheet with the slurry that is used for this transfer sheet, consider porosity and transfer sheet and TLC plateBetween suitable adhesiveness, the particle diameter of described carrier be preferably 0.1 to 100 μ m, more preferably 1 to 50 μ m and more more preferably 5 to40μm。
Consider that permission detects more accurately, in the time that the optic response by means of irradiating from ultraviolet ray detects spot,This transfer sheet preferably further contains fluorescence indicator. This fluorescence indicator can be known fluorescence indicator, and it can wrapDraw together magnesium tungstate for example, containing zinc silicate of manganese etc. This transfer sheet can be by glimmering with further adding this in slurry to transfer sheetLight indicator obtains. The content of this fluorescence indicator can be selected in the scope that allows to separate this target substance, and phaseBe generally 0.1 to 5 mass parts for the described carrier of 100 mass parts.
When by means of detecting when spot from the optic response that uses colour reagent, relate to colour reagent and color development treatmentKnown technology can be applicable to this transfer sheet. This colour reagent can comprise for example anisaldehyde solution, phosphorus molybdenum acid solution, iodine, indenesTriketone solution, chameleon solution, DNPH solution, manganese chloride solution and bromocresol green solution. Color development treatment can for example comprise and relating toAnd by spread, spray or expose colour reagent is attached in transfer sheet and this transfer sheet of optional heat with locating of developing the colorReason.
Consider the spot detecting more accurately in separating medium layer, preferably spot detection external member of the present invention enters oneStep comprises the aligning guide for TLC plate is aimed at predetermined relative location place with transfer sheet. This aligning guide can comprise exampleAs covered the position of this transfer sheet and prevent the mechanism that it moves in the horizontal direction for limiting TLC plate. More specifically, shouldAligning guide can comprise for by TLC plate in its end by chain connection to the mechanism in transfer sheet, for by TLC plateWith the framework that the transfer sheet covering fixedly secures in the horizontal direction, the projecting part providing in TLC plate or transfer sheet (asStake) and the depression parts (as hole) that provide on another part, and allow TLC plate and separating of transfer sheet bonding bondingDevice, as adhesive tape or ditch and ring fastener.
Spot detection method of the present invention can be undertaken by above-mentioned spot detection external member of the present invention.
Spot detection method of the present invention comprises the target substance in sample is deployed in the separating medium layer of TLC plateFirst step, covers the second step on separating medium layer in the position that at least this target substance has launched by this transfer sheetSuddenly, with shifting the capped separating medium layer of wet with solvent and the third step of transfer sheet, from covering separating medium layerTransfer sheet one side makes this transfer solvent evaporates to the spot on separating medium layer is transferred to the 4th step of transfer sheet, and lightLearn the 5th step that detects the spot that is transferred to transfer sheet.
First step can carry out in the mode that is similar to conventional TLC. That is to say, first step can be by dividingEdge's point sample from dielectric layer carries out, and the spot of this sample of optionally drying will separate Jie with the edge of point sampleMatter layer is immersed in mobile phase and the target substance in sample is deployed in separating medium layer. This target substance is to separate JieIn matter layer, show the material of the optic response identical with separating medium layer.
In second step, the position of having launched in separating medium layer at this target substance covers this transfer sheetCan. This transfer sheet can be covered in a part for separating medium layer, as the part that mobile phase has launched, still, considers fairPermitted to detect the position of spot, preferably covered on whole separating medium layer. Consider and allow to detect more accurately separating medium layerThe position of middle spot, second step is preferably in the step that covers this transfer sheet with respect to the pre-position of TLC plate.
Third step can be undertaken by following means: by transfer sheet one side covering on separating medium layer, this is turnedMove solvent and spray in whole transfer sheet, or transfer sheet and separating medium layer are immersed in transfer solvent, or use before coveringShift wet with solvent this transfer sheet to make after covering this separating medium layer enough moistening.
This transfer solvent can be to allow wash-out at least a portion from separating medium layer to form spot in separating medium layerThe solvent of component. This transfer solvent can be above-mentioned mobile phase, or known organic solvent or be selected from known organicThe mixture of two or more of solvent, water, bronsted lowry acids and bases bronsted lowry. Consider the solubility of target substance and the wash-out energy of target substancePower, this transfer solvent can be alcohol, as ethanol, methyl alcohol and 2-propyl alcohol, THF, ethyl acetate, chloroform or acetone. When this point of preparationFrom dielectric layer to such an extent as to while becoming the coating material being made up of the carrier of fixing polysaccharide derivates separating medium, this transfer solvent is excellentChoosing is alcohol, to make the polysaccharide derivates separating medium on carrier not dissolved.
Consider that shifting solvent is transferred sheet absorption with suitable speed, the 4th step is preferably passed through from transfer sheet one side airThe dry assorted transfer sheet covering on separating medium layer is carried out, and considers raising transfer amount, uses the cold sky of 10 to 30 DEG CIt is preferred that gas carries out air dry. This air is dry can carry out on whole transfer blade, or has therein turning of spotMove in the part of plate and carry out.
Can in the time that transfer sheet covers on separating medium plate or after dismounting transfer sheet, carry out the 5th step. The 5thIn step, this target substance shows the detectable optic response that is different from transfer sheet. As long as transfer sheet is to separating medium plateContact-making surface be table side (upperside), within the scope of the optic response of point that can obtain transfer, the light in the 5th stepLearn and detect and can carry out in the table side of transfer sheet or at dorsal part (reverseside).
Spot detection method of the present invention can comprise other step in the scope that can obtain effect of the present invention. ItsIts step has been deployed into separation Jie on it by target substance before can being included in transfer sheet being covered in to separating medium layerThe step that matter layer is dry. Consider the position that allows to detect more accurately spot in separating medium layer, preferably include this dry stepSuddenly. Consider and improve operability, this drying steps preferably uses the air drying steps of the hot-air of 20 to 80 DEG C, considersTo security, this step is preferably used the air drying steps of cold air.
Spot detection method of the present invention can be carried out by the following method, and for example as shown in fig. 1, preparation contains polysaccharideDerivative is applied to the TLC plate (A in Fig. 1) of the silica gel separating medium layer on it, will be such as racemic optical siomerism liquid solutionAn edge of the separating medium layer of sample spot in TLC plate, by the optical isomer in sample by this edge towards separatelyLaunch on one side, be used for the cold air dry (B in Fig. 1) of self-desiccation machine, will cover in TLC plate such as the transfer sheet of silica gel pieceSeparating medium layer on (C in Fig. 1), the transfer solvent such as ethanol is sprayed on the dorsal part of transfer sheet of covering to useShift this transfer sheet of wet with solvent and this separating medium layer (D in Fig. 1), be used for the cold sky of self-desiccation machine by the dorsal part of transfer sheetGas is dry this transfer sheet and the separating medium layer (E in Fig. 1) covering thereon gradually, and irradiates the dorsal part of this transfer sheet with UVTransfer to the spot (F in Fig. 1) in transfer sheet with optical detection.
The optical detection of the spot in the transfer sheet in the present invention can be used for observing the separating resulting on TLC plate. According to thisInvention, the spot of raffinate (raffinate) component and extracted component, as long as they on TLC plate, comprise TLC, separation is ledIts position relationship causing all can accurately detect. Further according to the present invention, when sample order point sample exhibition simultaneously on TLC plateWhile opening, can accurately detect the separation separately of multiple samples. This target substance can also be contained and be transferred to transfer sheet by collectionOn particular spots part and carry out extraction procedures and separate. In addition, this target substance can also be by collecting corresponding to containingThere is the separating medium layer segment of the part of the particular spots detecting in transfer sheet to separate.
Embodiment
[confirmation of Rf value]
First, by the inserts of 4.00 grams of CHIRALPAKIA (DaicelCorporation) (below also referred to as" IA inserts "), 0.60 gram of gypsum, the CMC(carboxymethyl cellulose of 4.00 gram 2%) 1110(DaicelCorporation) waterSnowtexC(NissanChemicalIndustries, the Ltd. of solution and 0.60 gram 20%) aqueous solution adds 0.40 toIn the mixed solution of gram water and 1.60 grams of ethanol, and fully stir to prepare the first slurry applying in ultrasonic. Meanwhile, will4.00 grams of silica gel (for liquid chromatography, from DaisoCo., Ltd., IR-60-5/20-U), 0.20 gram of gypsum, 6.00Gram 2% CMC(carboxymethyl cellulose) 1110(DaicelCorporation) aqueous solution and 0.04 gram of manganese and silicon containing acid zinc adds toIn the mixed solution of 2.02 grams of water and 2.80 grams of ethanol, and fully stir to prepare the second slurry applying in ultrasonic. MakeWith the spreader of preparing for TLC plate, these slurries are evenly applied in to the glass plate that demarcation strip is housed, by sprawling of slurryLayer air at 60 DEG C is dried and vacuum drying 3 hours, vacuumizes to prepare 12 simultaneously contain separately the first slurry with vavuum pumpThe first separating medium layer of material and the TLC that adjoins the second separating medium layer that is provided to the second slurry on the first separating medium layerPlate 1.
This first separating medium layer has the width of 2 centimetres, the length of 10 centimetres and the thickness of 150 μ m, this second separationDielectric layer has the width of 3 centimetres, the length of 10 centimetres and the thickness of 110 μ m. The first separating medium layer is corresponding to IA insertsLayer, the second separating medium layer is corresponding to silica gel layer. This IA inserts has the average grain diameter of 20 μ m, and silica gel has 14.4 μ m'sAverage grain diameter.
1% solution (about 1 μ L) by the racemic material of Troeger alkali (TB) in ethyl acetate is in distance longitudinallyThe about 1.0 centimeters points in bottom are on TLC plate 1. This TLC plate 1 is placed on and is launched in container, and this container comprises mixed solvent,Described mixed solvent contains n-hexane and ethanol with the volume ratio of 9:1, sample point under, make the Troeger alkali in sampleOptical isomer along longitudinal expansion of the first separating medium layer. After launching for the first time, with this TLC of cold air primary dryingPlate 1. Subsequently this TLC plate 1 is placed in the expansion container that contains ethanol, the first separating medium layer under, make Troeger alkaliOptical isomer be further expanded to the second separating medium layer by the first separating medium layer.
After launching for the second time, be dried and use ultraviolet ray to irradiate TLC plate 1 use cold air. As a result, at the second separating mediumIn layer, observe with green spot form the raffinate component that is moved to the second separating medium layer by the first separating medium layer separatelyThe spot (Fig. 2) of RTB and extracted component ETB. This first separating medium layer absorbs ultraviolet ray, and is seen as black region thusExamine. Spot on the position being arrived by the position of point sample on the first separating medium layer, developing solution and the second separating medium layerCenter determine and the Rf value of this spot show that the raffinate component of Troeger alkali and extracted component have respectively about 0.5With about 0.35 Rf value.
1% solution (about 1 μ L) by the racemic material of flavanones (FLV) in ethyl acetate is with above-mentioned similar fashionPoint is on another piece TLC plate 1, and the racemic material of flavanones launches in the second separating medium layer, observes in the second separation and is situated betweenThe position of the spot of raffinate component RFLV and extracted component EFLV in matter layer. As a result, two kinds of components are all with the second separating mediumGreen spot form on layer is observed (Fig. 3). The raffinate component of flavanones and extracted component have respectively about 0.3 HeAbout 1.75 Rf value.
[embodiment 1]
Then, use the spreader of preparing for TLC plate that the first slurry is applied to glass pane surface equably, will starchMaterial sprawl layer of air dry and at 60 DEG C, vacuumize with vavuum pump in vacuum drying 3 hours, only contain the to prepareThe first separating medium layer of one slurry also has the TLC plate 2 of the thickness of the width of 2 centimetres, the length of 10 centimetres and 150 μ m.
1% solution (about 1 μ L) and flavanones by the racemic material of Troeger alkali (TB) in ethyl acetate(FLV) 1% solution (about 1 μ L) of racemic material in ethyl acetate is longitudinally apart from about 1.0 centimeters in bottomPut respectively on TLC plate 2. This TLC plate 2 is placed on and is launched in container, and this container comprises mixed solvent, described mixed solvent withThe volume ratio of 9:1 contains n-hexane and ethanol, sample point under, make two samples simultaneously along longitudinal exhibition of TLC plate 2Open. After expansion, by the dry TLC plate 2 use cold airs expansion with termination spot.
On the first separating medium layer of this TLC plate 2, cover the silica gel piece with same size of rectangular shape, by ethanolSpray on the silica gel piece of whole covering to soak this silica gel piece and this first separating medium layer, silica gel piece cover this firstOn separating medium layer time, it is applied to cold air to be dried this first separating medium layer and this silica gel piece, and make thus firstSpot on separating medium layer is transferred on this silica gel piece by the first separating medium layer.
By the second slurry being applied on glass plate, being dried, peeling off shape thus from glass plate with above-mentioned similar fashionThe thickness becoming is the second separating medium layer of about 300 μ m and regulates this layer to have rectangular shape, obtains thus silica gel piece.
Once ethanol is dry, and silica gel piece is separated with TLC plate 2, and the contact to TLC plate 2 with ultraviolet ray irradiation silica gel piecePlane. As a result, the raffinate component RTB of Troeger alkali and flavanones and RFLV and extracted component ETB and EFLV are respectively with greenSpot is detected (Fig. 4).
[reference example]
Prepare the 3rd slurry in the mode identical with preparation the first slurry, except using CHIRALPAKIC (DaicelCorporation) inserts (below also referred to as " IC inserts ") replaces IA inserts. With the side identical with preparation TLC plate 2Formula is prepared TLC plate 3, and except using the 3rd slurry, it only contains the 3rd separating medium layer of the 3rd slurry. By Troeger alkali(TB) the racemic material of 1% solution (about 1 μ L) of racemic material in ethyl acetate and flavanones (FLV) is at acetic acid1% solution in ethyl ester with on TLC plate 2 in launch identical mode and launch on TLC plate 3 simultaneously, and spot is turnedMove on on silica gel piece. Even if be known that this inserts can not separate these with the combinations thereof of mixed solvent in the time using postOptical isomer in sample.
Once ethanol is dry, and silica gel piece is separated with TLC plate 3, and the contact to TLC plate 3 with ultraviolet ray irradiation silica gel piecePlane. As a result, Troeger alkali and flavanones detect as the form of single green spot STB and SFLV respectively, and not pointSeparate (Fig. 5).
Industrial applicability
TLC has been conventionally used as the research separation condition of column chromatography and the means of isolation of target substances. With prior artCompare, in the time that use is difficult to detect by means of optic response the separating medium separating thus, the present invention can detect more accuratelyThe separation of target substance. Therefore, expection the present invention contributes to further expand the application of this type of separating medium and further developUse separation and the purification technique of this type of separating medium.
Description of reference numerals
The each state of A to F in exemplary spot detection procedure of the present invention
The spot of the raffinate component of RTBTroeger alkali
The spot of the extracted component of ETBTroeger alkali
The spot of the raffinate component of RFLV flavanones
The spot of the extracted component of EFLV flavanones
The spot of the racemic material of STBTroeger alkali
The spot of the racemic material of SFLV flavanones
Claims (5)
1. spot detection external member, comprises and has the TLC plate of separating medium layer and will cover on described separating medium layer with by instituteThe spot of stating on separating medium layer is transferred to the transfer sheet on it,
The separating medium that wherein forms described separating medium layer is polysaccharide derivates, and
Described transfer sheet has and is different from the optic response of described separating medium layer and also has a porous,
Described transfer sheet is silica gel piece.
2. spot detection external member as claimed in claim 1, further comprise for by described TLC plate and described transfer sheet in advanceThe aligning guide that fixed relative position place is aimed at.
3. the spot detection of implementing in thin-layered chromatography by using spot detection external member as claimed in claim 1 or 2Method,
The method comprises:
Optical isomer in sample is expanded to the step in the described separating medium layer of described TLC plate, this step byEdge's point sample of separating medium layer carries out, and as below, separating medium layer is immersed in mobile taking the edge of point sampleMutually and the target substance in sample is deployed in separating medium layer;
The position of having launched at least described optical isomer covers described transfer sheet on described separating medium layerStep;
With shifting the capped separating medium layer of wet with solvent and the step of transfer sheet;
Make described transfer solvent evaporates so that described separation is situated between from described transfer sheet one side covering described separating medium layerSpot on matter layer is transferred to the step in described transfer sheet;
Optical detection is transferred to the step of the spot of described transfer sheet,
The separating medium that forms described separating medium layer is polysaccharide derivates.
4. spot detection method as claimed in claim 3, is further included in described transfer sheet is covered to described separation JieBefore on matter layer, dry described target substance has been expanded to the step of separating medium layer wherein.
5. the spot detection method as described in claim 3 or 4, wherein said transfer sheet covers at predetermined relative position placeOn described TLC plate.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011147664 | 2011-07-01 | ||
| JP2011-147664 | 2011-07-01 | ||
| PCT/JP2012/066563 WO2013005642A1 (en) | 2011-07-01 | 2012-06-28 | Spot detection set, spot detection method, and transfer-receiving sheet |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103649746A CN103649746A (en) | 2014-03-19 |
| CN103649746B true CN103649746B (en) | 2016-05-18 |
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|---|---|---|---|
| CN201280032952.0A Active CN103649746B (en) | 2011-07-01 | 2012-06-28 | Spot detection external member, spot detection method and transfer sheet |
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| Country | Link |
|---|---|
| JP (1) | JP6043718B2 (en) |
| CN (1) | CN103649746B (en) |
| DE (1) | DE112012002794B4 (en) |
| IN (1) | IN2014CN00680A (en) |
| WO (1) | WO2013005642A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104792914A (en) * | 2015-04-03 | 2015-07-22 | 广东医学院 | Plane chromatography and micro-porous plate array mapping correlation experiment method and application thereof |
| US20190170713A1 (en) * | 2016-12-26 | 2019-06-06 | Panasonic Intellectual Property Management Co., Ltd. | Thin layer chromatography plate and sample analysis method using same |
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| JPH0743364B2 (en) * | 1990-03-23 | 1995-05-15 | 株式会社ピーシーシーテクノロジー | Analysis method for cardiac glycosides by immunoassay |
| US5877028A (en) | 1991-05-29 | 1999-03-02 | Smithkline Diagnostics, Inc. | Immunochromatographic assay device |
| JP3086812B2 (en) * | 1994-02-24 | 2000-09-11 | アトー株式会社 | Method for transferring fat-soluble compound from fixed layer to resin film |
| JP4320068B2 (en) * | 1997-10-03 | 2009-08-26 | ダイセル化学工業株式会社 | Separating agent for optical isomers and process for producing the same |
| DE60222243T2 (en) * | 2001-03-07 | 2008-01-03 | Daicel Chemical Industries, Ltd., Sakai | RELEASE AGENT FOR OPTICAL ISOMER |
| CN100364661C (en) * | 2003-04-24 | 2008-01-30 | 大赛璐化学工业株式会社 | Agent for separating optical isomers |
| US20100059442A1 (en) | 2006-11-17 | 2010-03-11 | The Regents Of The University Of California | Method and system for separating analytes |
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- 2012-06-28 WO PCT/JP2012/066563 patent/WO2013005642A1/en active Application Filing
- 2012-06-28 DE DE112012002794.9T patent/DE112012002794B4/en active Active
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| US5306645A (en) * | 1990-01-31 | 1994-04-26 | Shimadzu Corporation | Concentration and transfer methods for a chromatogram and an LC/IR measuring method |
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Also Published As
| Publication number | Publication date |
|---|---|
| IN2014CN00680A (en) | 2015-04-03 |
| DE112012002794T5 (en) | 2014-03-20 |
| DE112012002794B4 (en) | 2023-11-09 |
| CN103649746A (en) | 2014-03-19 |
| WO2013005642A1 (en) | 2013-01-10 |
| JPWO2013005642A1 (en) | 2015-02-23 |
| JP6043718B2 (en) | 2016-12-14 |
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