CN1715910A - Microminiaturized liquid phase chromatography separating method - Google Patents
Microminiaturized liquid phase chromatography separating method Download PDFInfo
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- CN1715910A CN1715910A CN 200510057132 CN200510057132A CN1715910A CN 1715910 A CN1715910 A CN 1715910A CN 200510057132 CN200510057132 CN 200510057132 CN 200510057132 A CN200510057132 A CN 200510057132A CN 1715910 A CN1715910 A CN 1715910A
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- microchannel
- liquid phase
- phase chromatography
- microminiaturization
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Abstract
The microminiaturized liquid chromatographic separation method features that the method adopts dry filled capillary column or filled micro channel as separating channel and capillary effect as driving force in realizing sample separation. The present invention needs no additional driving device, and has the features of low cost, fast speed, high separation efficiency, high detection sensitivity and micro size.
Description
Technical field
The present invention relates to a kind of method for separating liquid phase chromatography, the method for separating liquid phase chromatography of a kind of microminiaturization specifically.
Background technology
Since Izmailor in 1938 and Schraiber had separated composition in the various plants tincture first with thin-layered chromatography since, thin-layer chromatography (TLC) had occupied critical role in compartment analysis.Thin-layer chromatography is coated on adsorbent on the thin layer plate, behind the point sample thin layer plate is immersed developping agent, and sample component is launched on thin layer plate under capillary action drives, and reaches separation.Thin-layer chromatography has advantages such as equipment is simple, easy to operate, velocity of separation is fast, detection mode is versatile and flexible.Particularly thin-layer chromatography relies on capillary action to drive---and do not need to add driving force, therefore can be called " self-driven ", the isolation technics equipment of this " self-driven " is simple, and is very tempting aspect miniaturization.But the adsorbent consumption is big in the thin-layer chromatography of prior art, adopts generally that particle is big, the adsorbent (being generally silica gel or aluminium oxide) of particle size range broad, and it is not high to cause post to be imitated, and has limited its range of application.Simultaneously, in thin-layer chromatography, there is serious horizontal broadening in sample point, has reduced detection sensitivity.And thin-layer chromatography also is difficult to further miniaturization as planar chromatograph.
Summary of the invention
The object of the present invention is to provide a kind of method for separating liquid phase chromatography with microminiaturized characteristics.
The object of the present invention is achieved like this: a kind of method for separating liquid phase chromatography of microminiaturization is characterized in that: it is with the packed capillary column of drying or to fill the microchannel be split tunnel, is that driving force realizes sample separation with the capillary action.
The present invention adopts homogenate method or situ aggregation method to prepare packed capillary column or fills the microchannel; Capillary column one end of filling is immersed in the sample solution, extract rapidly then, realize sample introduction, again packed capillary column sample introduction end is immersed and launch solvent, or be the point sample kapillary with the kapillary of fine inner diameter, the direct point sample in aperture place on chip, the aperture place on chip adds the expansion solvent then; Launching solvent drive sample flow under capillarity drives crosses the filler in capillary column or the microchannel and obtains separation.
Above-mentioned employing homogenate method packed capillary column, its concrete steps are: at first quartz capillary one end is sealed (if kapillary has coating, then need to remove earlier coating), then filler is dissolved in acetone, make the homogenate of concentration about 10%, place the homogenate pond, homogenate pond one end connects quartz capillary, the other end connects high-pressure pump, and high-pressure pump pressure is brought up to 30Mpa, and homogenate is pressed in the quartz capillary, filler is decompressed to normal pressure after being full of kapillary gradually, take off capillary column and be cut into segment, wherein solvent evaporates is done promptly get packed capillary column, filled capillary pipe put into the expansion solvent vapor chamber embezzle close standby.
Above-mentioned filler is the high performance liquid chromatography filler special, and particle diameter is 3～5 μ m.
Above-mentioned filling microchannel, be to be chip with the light transmissive material, chip is provided with the microchannel, and the cover plate that is with holes of bonding thereon, the position of aperture is aimed at the two ends of microchannel on the chip respectively on the cover plate, and the microchannel of adopting situ aggregation method to fill on the chip again makes.
The filling microchannel that the present invention adopts situ aggregation method to make, its concrete steps are: the polymeric solution for preparing is splashed in the aperture on the cover plate of filling the microchannel, polymeric solution is full of the microchannel under the capillarity effect; Bring out polyreaction with the UV-irradiation microchannel, after reaction finishes, use washed with methanol, dry and get final product.
Said chip or be glass or dimethyl silicone polymer PDMS or polymetylmethacrylate; Said chip is provided with one or more microchannels.
The said chip size is 50mm * 30mm * 3mm; Above-mentioned bonding adopt hot key and or chemical bond and.
Above-mentioned polymeric solution is made up of polymer monomer, crosslinking chemical, initiating agent, solvent, pore-foaming agent, specifically by polymer monomer butyl acrylate 23.6%, crosslinking chemical ethylene glycol dimethacrylate 16%, azoisobutyronitrile 0.4%, ethanol 30%, methyl alcohol 30% is formed.
The internal diameter of above-mentioned capillary column or microchannel is 0.05～1.0mm, the packed capillary column that is prepared into or fill the microchannel and be equal once integral post.
The present invention is based on the thin-layer chromatography principle, with the filled capillary pipe of drying or to fill the microchannel be split tunnel, utilizes capillary action to realize chromatographic resolution for driving force.The present invention is by the preparation of fine inner diameter packed capillary column and prepare integral post in chip microchannel, has realized comprising the microminiaturization of systems such as post preparation, sample introduction, separation, detection.Owing to adopted high-quality filler, improved separation efficiency in the present invention, velocity of separation is very fast, can finish separation in 1-3 minute usually, and its resolution is much higher than thin-layer chromatography, and is suitable with HPLC; Simultaneously, because the restriction of capillary wall or microchannel, there is not horizontal broadening in sample point, has improved detection sensitivity, and under the same terms, the remolding sensitivity thin-layer chromatography is high more than 5 times.The present invention is again a kind of microminiaturized method simultaneously, and sample size is about 10
-9The L level, the developping agent demand is less than 10
-4L has saved reagent, has reduced cost.In addition, owing to do not need additional drive system, equipment is simple, and is easy to use in the present invention.
Description of drawings
Fig. 1 is the structural representation of packed capillary column in the embodiment of the invention 1.
Fig. 2 is the expanding unit of the embodiment of the invention 1.
Fig. 3 is a laser-induced fluorescence (LIF) scanning detection architecture synoptic diagram among the present invention.
Fig. 4 fills the Micro Channel Architecture synoptic diagram on chip in the embodiment of the invention 2.
Embodiment
Embodiment 1:
Post preparation: adopt the homogenate legal system to be equipped with packed capillary column.Getting internal diameter is 0.05-1.0mm, is about the quartz capillary 1 of 30cm, knocks in a bit of silica gel (being about 2mm, silica gel diameter 5 μ m) in one port, makes stopper (this stopper can not pass through filler by solvent) in about 10 seconds at 600 ℃ of left and right sides sintering; With the whole burning-offs of capillary coating.Be that silica gel or the ODS silica gel of 3 μ m or 5 μ m is that filler 2 is dissolved in acetone with particle diameter then, make the homogenate of concentration about 10%, place the homogenate pond.Homogenate pond one end connects kapillary, the other end connects high-pressure pump, high-pressure pump pressure is brought up to 30Mpa, homogenate is pressed in the kapillary, filler is decompressed to normal pressure after being full of kapillary gradually, take off kapillary and be cut into the long segment of 3cm, solvent evaporates is wherein done promptly obtaining packed capillary column 3, put into launch solvent vapor chamber embezzle close standby.
Sample introduction: get above-mentioned packed capillary column, the one end is immersed in the sample solution, extract rapidly again, just can realize sample introduction.
Separate: packed capillary column 3 sample introduction ends are immersed launch solvent 4.Launch solvent 4 and drive sample flow cross filler in the packed capillary column under capillarity drives, different component in the sample is owing to the absorption on filler, distribute the difference of behavior, the development rate difference, and obtain separating.The activity of polarity per sample, solubleness and adsorbent can be selected different expansion solvents for use.Detachment process can be finished at 2-3 minute usually, and development distance is about 20mm.
Detect: detection can adopt scanning to detect, but also carries out graphical analysis after the direct imaging.
Scanning detects: with the laser-induced fluorescence (LIF) is example.Laser with 532nm is that exciting light 5 focuses on the packed capillary column, and simultaneously, packed capillary column is at the uniform velocity mobile vertically under stepper motor 6 drives, and can finish the scanning to packed capillary column.Composition in the packed capillary column produces fluorescence signal 7 under laser excitation, this signal can be converted into electric signal and record with electrooptical device.The fluorescence signal that component in the sample produces is different with the fluorescence signal that launches solvent, thereby can distinguish the sample component development distance; Simultaneously, because the power of fluorescence signal is relevant with concentration of component, can this carry out quantitative test.
Adopt scanning to detect, need derive the material that does not have fluorescence signal; Also available the silica gel of fluorescence is arranged is filler, separates back display dot under UV-irradiation.
Above-mentioned scanning detects also can adopt other light analytical approach such as modes such as scattering, absorption to replace laser-induced fluorescence (LIF) to carry out scanning analysis.
Imaging analysis detects: sample adopts image device (as CCD, CMOS) imaging after launching to separate, and it is qualitative and quantitative to adopt software to carry out then.The imaging analysis device is simple, and speed is fast.
Embodiment 2:
Fill the preparation of microchannel: be starting material at first, be processed into the chip 8 that is of a size of 50mm * 30mm * 3mm with glass.On chip, process 3 microchannels 9 then, the wide about 0.1-1mm in each microchannel, dark 0.05-0.5mm is about 3cm.Again bonding (adopt hot key and or chemical bond and) on be equipped with the cover plate 11 of 6 about 5mm apertures 10 of diameter, the position of 6 apertures is aimed at 6 ports at two ends, 3 microchannels respectively on the cover plate.
Situ aggregation method is adopted in the filling of chip microchannel.After the steps include: key and finishing, with polymeric solution (polymer monomer butyl acrylate 23.6%, the crosslinking chemical ethylene glycol dimethacrylate 16% for preparing, azoisobutyronitrile 0.4%, ethanol 30%, methyl alcohol 30%) splash in the aperture of each microchannel one end on the chip, solution is full of the microchannel under the capillarity effect.Bring out polyreaction with the UV-irradiation microchannel.After 4 hours, use washed with methanol, dry and get final product.
Sample introduction: the kapillary (25-50 μ m) with fine inner diameter is the point sample kapillary, the direct point sample in aperture place on chip.
Separate: the aperture place on chip adds developping agent, and developping agent drives sample flow and crosses filler in the microchannel under capillarity drives, and different component in the sample is owing to the absorption on filler, distribute the difference of behavior, the development rate difference, and obtain separating.This process can be finished at 2-3 minute, and development distance is about 20mm.
Detect: with embodiment 1.
Claims (10)
1, a kind of method for separating liquid phase chromatography of microminiaturization is characterized in that: it is with the packed capillary column of drying or to fill the microchannel be split tunnel, is that driving force realizes sample separation with the capillary action.
2, the method for separating liquid phase chromatography of microminiaturization as claimed in claim 1 is characterized in that: adopt homogenate method or situ aggregation method to prepare packed capillary column or filling microchannel; Capillary column one end of filling is immersed in the sample solution, extract rapidly then, realize sample introduction, again packed capillary column sample introduction end is immersed and launch solvent, or be the point sample kapillary with the kapillary of fine inner diameter, the direct point sample in aperture place on chip, the aperture place on chip adds the expansion solvent then; Launching solvent drive sample flow under capillarity drives crosses the filler in capillary column or the microchannel and obtains separation.
3, the method for separating liquid phase chromatography of microminiaturization as claimed in claim 2, it is characterized in that: adopt homogenate method packed capillary column, its concrete steps are: at first quartz capillary one end is sealed, then filler is dissolved in acetone, make the homogenate of concentration 8-12%, place the homogenate pond, homogenate pond one end connects quartz capillary, the other end connects high-pressure pump, and high-pressure pump pressure is brought up to 30Mpa, and homogenate is pressed in the quartz capillary, filler is decompressed to normal pressure after being full of kapillary gradually, take off kapillary and be cut into segment, wherein solvent evaporates is done promptly obtaining filled capillary pipe, filled capillary pipe put into launch solvent vapor chamber embezzle close standby.
4, the method for separating liquid phase chromatography of microminiaturization as claimed in claim 3 is characterized in that: described filler is the high performance liquid chromatography filler special, and particle diameter is 3～5 μ m.
5, the method for separating liquid phase chromatography of microminiaturization as claimed in claim 1 or 2, it is characterized in that: described filling microchannel, be to be chip with the light transmissive material, chip is provided with the microchannel, and the cover plate that is with holes of bonding thereon, the position of aperture is aimed at the two ends of microchannel on the chip respectively on the cover plate, and the microchannel of adopting situ aggregation method to fill on the chip again makes.
6, the method for separating liquid phase chromatography of microminiaturization as claimed in claim 5, it is characterized in that: the filling microchannel of adopting situ aggregation method to make, its concrete steps are: the polymeric solution for preparing is splashed in the aperture on the cover plate of filling the microchannel, polymeric solution is full of the microchannel under the capillarity effect; Bring out polyreaction with the UV-irradiation microchannel, after reaction finishes, use washed with methanol, dry and get final product.
7, the method for separating liquid phase chromatography of microminiaturization as claimed in claim 5 is characterized in that: described chip or for glass or dimethyl silicone polymer or polymethylmethacrylate; Described chip is provided with one or more microchannels.
8, the method for separating liquid phase chromatography of microminiaturization as claimed in claim 5 is characterized in that: described die size is 50mm * 30mm * 3mm; Described bonding adopt hot key and or chemical bond and.
9, the method for separating liquid phase chromatography of microminiaturization as claimed in claim 6 is characterized in that: described polymeric solution is by polymer monomer, crosslinking chemical, initiating agent, solvent, and pore-foaming agent is formed.
10, as the method for separating liquid phase chromatography of claim 1,2 or 3 described microminiaturizations, it is characterized in that: the internal diameter of described capillary column or microchannel is 0.05～1.0mm, the packed capillary column that is prepared into or fill the microchannel and be equal once integral post.
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| CN2005100571329A CN100406886C (en) | 2005-06-22 | 2005-06-22 | Microminiaturized liquid phase chromatography separating method |
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| CN2005100571329A CN100406886C (en) | 2005-06-22 | 2005-06-22 | Microminiaturized liquid phase chromatography separating method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103180726A (en) * | 2010-06-25 | 2013-06-26 | 帝国革新有限公司 | Miniature hplc device |
| CN104297025A (en) * | 2014-09-10 | 2015-01-21 | 閤康生物科技股份有限公司 | Liquid sample drying device, dried sample test piece and preparation method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4520621B2 (en) * | 2000-11-01 | 2010-08-11 | 信和化工株式会社 | Chromatographic separation column, solid phase extraction medium, and chromatographic sample injection system |
| DE10140680A1 (en) * | 2001-08-24 | 2003-03-06 | Bayer Ag | Spectroscopic test system based on microcapillaries |
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2005
- 2005-06-22 CN CN2005100571329A patent/CN100406886C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103180726A (en) * | 2010-06-25 | 2013-06-26 | 帝国革新有限公司 | Miniature hplc device |
| CN103180726B (en) * | 2010-06-25 | 2015-07-01 | 帝国革新有限公司 | Miniature HPLC device |
| CN104297025A (en) * | 2014-09-10 | 2015-01-21 | 閤康生物科技股份有限公司 | Liquid sample drying device, dried sample test piece and preparation method |
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| CN100406886C (en) | 2008-07-30 |
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Granted publication date: 20080730 |