CN103691318A - Micro-scale substance separating method and capillary column transverse eletrochromatography separating device - Google Patents
Micro-scale substance separating method and capillary column transverse eletrochromatography separating device Download PDFInfo
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- CN103691318A CN103691318A CN201410005331.4A CN201410005331A CN103691318A CN 103691318 A CN103691318 A CN 103691318A CN 201410005331 A CN201410005331 A CN 201410005331A CN 103691318 A CN103691318 A CN 103691318A
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Abstract
The invention discloses a micro-scale substance separating method and a capillary column transverse eletrochromatography separating device. The separating method comprises the step of introducing a sample into a separating column of a separating device, wherein the separating column is composed of a capillary tube which is used as an external electrode and a conductive material which is used as an internal electrode and penetrates through the capillary tube along the axial direction; the internal electrode and the external electrode are coaxial, and are respectively connected with the two ends of a power supply; the separating column transversely applies electric fields upwards, and electrophoresis effect is utilized for transversely transferring to-be-separated substances, so that contact probability of the to-be-separated substances and a column surface is increased, and separation is realized according to difference of interaction force sizes of the to-be-separated substances and the column surface; an absorption analyzing process of the to-be-separated substances and the column surface is completed by switching direction of the electric fields, so that resolution among the to-be-separated substances is improved. According to the invention, an electrophoretic technique and a chromatographic technique are integrated to realize effective separation of different substances, operation of the separating method and manufacturing of the special-purpose device are simple; and moreover, online detection can be realized.
Description
Technical field
The present invention relates to a kind of separation method and special equipment thereof of minute yardstick material, specifically refer to the separation method and the capillary column transverse electric chromatographic separation device that electrophoretic techniques and chromatographic technique are blended in to a kind of minute yardstick material of one.
Background technology
At present, minute yardstick material is as cell, large biological molecule, synthetic particle, colloid etc., and its Isolation and screening has become a preferred main research in each research field in the present age.Traditional isolation technics is difficult to the sample of size of separation wider range simultaneously, and is difficult to obtain the target substance of single size, and these all will be made troubles to follow-up study.For realizing the separation of minute yardstick material, some clog-free, non-filled-type piece-rate systems are established, and the overwhelming majority belongs to continuous type isolation technics, this type of technology has many advantages, such as can realize sample continuous isolation and screening, can realize the quick separation of a large amount of samples etc.Yet the equal more complicated of the apparatus structure of this type of technology and manufacturing process, and equipment making required precision is high, and for the separation of a plurality of components, device preparation is more difficult.Multiple exit design feature is difficult to realize the online detection of sample, so be not suitable for the compartment analysis of the unknown or complex sample.In addition, most of micro-isolation technics is compared with existing chromatographic technique, and its separating degree is all lower.Therefore, in continuous type isolation technics actual application, must face some challenges.Based on above limitation, for better studying minute yardstick material, need to develop all better simply technology of a kind of high-resolution, device making and experimental implementation.What is more important, requires technology can easily realize the online detection of sample.
Summary of the invention
The object of the invention is in order to overcome the low deficiency of minute yardstick separating substances degree in non-filled-type piece-rate system, and provide a kind of separation method and special equipment capillary column transverse electric chromatographic separation device thereof of minute yardstick material, the present invention transversely introduces electric field at the splitter of separator, and chromatographic effect is introduced on post surface, after entering splitter, can under electric field action, there is transverse electric migration in minute yardstick material, by the electromigration behavior of material, increase the contact probability on particle and post surface, and then the contribution that improves chromatographic effect, finally reach the object that improves separating degree
For solving the problems of the technologies described above, technical scheme of the present invention is as follows:
A separation method for minute yardstick material, comprises the following steps:
1. sample is introduced in injector by micro syringe;
2. open the power switch of capillary column transverse electric chromatographic separation device, make the splitter of capillary column transverse electric chromatographic separation device transversely form electric field;
3. sample enters described splitter by sampling valve under the drive by transport medium, under additional transverse electric field effect, in sample there is lateral transfer in material to be separated in splitter, and when material to be separated moves to post surface, material to be separated can interact with post surface;
4. the switching of power positive cathode direction, make material to be separated move back and move in splitter inner transverse, and with splitter inner surface generation adsorption-desorption process, difference according to cross force causes distribution coefficient between material there are differences, thereby cause material streamwise to be separated to have different speed, realize the separation of different material;
5. separate substance enters in detector with different elution times, detects online.
Described electric field presentation mode is the pulse power or AC power.
A capillary column transverse electric chromatographic separation device, includes and provides the pump of transport medium, injector, detector, power supply and one end to be connected injector, the splitter of other end connection detector, and wherein said splitter includes:
A capillary, this capillary is as external electrode;
A conductive material, this conductive material is as interior electrode, runs through vertically capillary and in capillary center, in electrode with coaxial with external electrode, interior electrode be connected both ends of power with external electrode.
Described conductive material is platinum filament, spun gold or filamentary silver.
Separating mechanism of the present invention: material to be separated is after sampling valve is introduced splitter, in splitter, it is electrophoretic techniques that material to be separated will be subject to transverse electric field effect generation transverse shifting, when material to be separated moves to post surface, it is chromatographic technique that material to be separated and post surface interact generation.In the present invention, material to be separated is because of self property difference, its suffered horizontal force will produce difference, and then make material lateral transfer to be separated apart from producing difference, finally cause material to be separated and post surface action power there are differences, be that the distribution coefficient of material to be separated in splitter of the present invention there are differences, finally realize the separation and analysis of material to be separated.In system of the present invention, utilize the switching of direction of an electric field, make material to be separated transversely have transition process repeatedly at splitter, and then make there is adsorption-desorption process between material to be separated and post surface, by changing the frequency of electric field, improve theoretical cam curve, and then improve the separating degree of clog-free, non-filled-type piece-rate system.
Above-mentioned self property comprises size or molecular weight, surface charge and diffusion coefficient; Above-mentioned horizontal force is electrophoretic force and viscous resistance; Above-mentioned material to be separated and the interaction between post surface can exist for ionic interaction (electrostatic interaction), apolar interaction or ionic interaction and apolar interaction simultaneously.
In a word, the present invention, by by the on-line coupling of splitter and detector, has overcome current micro-isolation technics the unknown or complicated minute yardstick material has been difficult to the difficulty that ON-LINE SEPARATION is analyzed.In addition, the present invention is effectively melted into a whole electrophoretic techniques and chromatographic technique, has greatly improved the theoretical cam curve of clog-free, non-filled-type splitter, and then realizes the efficient separation of sample, be expected to obtain the minute yardstick material of single size, for the follow-up study of minute yardstick material lays the foundation.
Accompanying drawing explanation
Fig. 1 is capillary column transverse electric chromatographic separation device schematic diagram of the present invention.
Fig. 2 is different size granules of polystyrene separating effect figure in the embodiment of the present invention.
Fig. 3 is different surfaces electric charge granules of polystyrene separating effect figure in the embodiment of the present invention.
The specific embodiment:
In Fig. 1,1, transport medium, 2, six-way injection valve, 3, sample, 4, waste liquid, 5, PEEK pipe, 6, conductive material-interior electrode, 7, capillary-external electrode, 8, power supply, 9, detector, 10, triple valve.
With reference to figure 1, a kind of capillary column transverse electric chromatographic separation device, include and provide the pump of transport medium, injector, detector, power supply and one end to be connected injector, the splitter of other end connection detector, wherein said splitter includes: a capillary 7, and this capillary 7 is as external electrode; A conductive material 6, described conductive material 6 can be platinum filament, spun gold or filamentary silver, this conductive material 6 is as interior electrode, run through vertically capillary 7, for guaranteeing that in splitter, the suffered transverse electric field force of material to be separated is identical everywhere, interior electrode must be placed in external electrode center, and interior electrode is with coaxial with external electrode, and connect respectively power supply 8 two ends, and then transversely produce electric field at splitter.The present invention is without the high-pressure pump that uses conventional liquid phase systems, injector adopts six-way injection valve 2, for material to be separated is introduced to splitter, and realize online and detecting, capillary 7 one end of splitter connect six-way injection valve 2 to introduce sample 3 by the triple valve 10 being fixedly installed, the other end is by triple valve 10 connection detectors 9 that are fixedly installed so that the online detection of real separate substance, and conductive material 6 two ends in capillary outside are accepted by PEEK pipe 5.
Utilize the capillary column transverse electric chromatographic separation device of above-mentioned special use and the separation method of disclosed a kind of minute yardstick material comprises the following steps:
1. sample 3 is introduced in six-way injection valve 2 by micro syringe;
2. the switch of opening power 8, makes splitter transversely form electric field;
3. sample 3 enters splitter by six-way injection valve 2, triple valve 10 under the drive of the transport medium 1 being provided by pump, and under additional transverse electric field effect, in sample, lateral transfer occurs material to be separated in splitter;
4. the cathode and anode directions of power supply 8 switches, make material to be separated migration back and forth in the horizontal, and with post surface, adsorption-desorption process occurs, the difference of cross force there are differences distribution coefficient between material, and then cause material streamwise to be separated to have different speed, realize the separation of different material;
5. separate substance is entered in detector 9 by triple valve 10 with different elution times, detects online effluent discharge 4 simultaneously.
In concrete separation operation process, described power supply is the pulse power or AC power.Described horizontal force is the interaction force between electrophoretic force, viscous resistance, material to be separated and post surface; Described material to be separated and the interaction between post surface can exist for ionic interaction (electrostatic interaction), apolar interaction or ionic interaction and apolar interaction simultaneously.
In capillary column transverse electric chromatographic system, when splitter is fixedly time, number of times is resolved in the absorption between material to be separated and post surface directly affects the separating degree between material to be separated.In capillary column transverse electric chromatographic technique, by optimizing the parameters such as electric field level (voltage and frequency), electrophoresis time, transport medium flow velocity, can improve the separating degree between material to be separated.Optimum results shows, extra electric field frequency
fand the duration
twith elution time
t ebecome positive correlation, voltage U pair
t enothing affects, but affects reservation amount and the peak shape of particle; In addition, voltage U and electric field duration
twith half-peak breadth
w 1/2become negative correlation,
fwith
w 1/2become positive correlation; In certain limit, transport medium flow rate effect is little.In the present invention, under electric field action, will there is lateral transfer in material to be separated, and because material lateral transfer to be separated there are differences the interaction force on itself and post surface apart from difference, distribution coefficient is different.In addition, in the present invention, adopt the variation of pulse frequency to make material to be separated and post surface that adsorption-desorption process occur.Based on above two reasons, finally realize the separation between the different material of minute yardstick.
The present invention realizes separated in actual separation process to minute yardstick material, the granules of polystyrene thing of take is evaluated its resolution ratio as example.Capillary column transverse electric chromatographic technique separation condition is as follows, and splitter length is 20 cm; Voltage is 1.2 V; Frequency is 0.5 Hz; Electric field application time is 10 min; Pulse period is respectively 0.4 and 0.6; Transport medium is distilled water; Flow velocity is 1.0 mL/h.As Fig. 2, Fig. 3.From Fig. 2,3, can draw, capillary column transverse electric chromatographic separation technology has been realized the separated object of different minute yardstick materials (different size or different surfaces electric charge).The invention discloses capillary column transverse electric chromatographic technique, effectively electrophoretic techniques and chromatographic technique are melted into a whole first, improve the resolution ratio between material to be separated, obtain the minute yardstick material of single size, and this technology can realize online detection.
Claims (4)
1. a separation method for minute yardstick material, comprises the following steps:
1. sample is introduced in injector by micro syringe;
2. open the power switch of capillary column transverse electric chromatographic separation device, make the splitter of capillary column transverse electric chromatographic separation device transversely form electric field;
3. sample enters described splitter by sampling valve under the drive by transport medium, under additional transverse electric field effect, in sample there is lateral transfer in material to be separated in splitter, and when material to be separated moves to post surface, material to be separated can interact with post surface;
4. the switching of power positive cathode direction, make material to be separated move back and move in splitter inner transverse, and with splitter inner surface generation adsorption-desorption process, difference according to cross force causes distribution coefficient between material there are differences, thereby cause material streamwise to be separated to have different speed, realize the separation of different material;
5. separate substance enters in detector with different elution times, detects online.
2. the separation method of a kind of minute yardstick material according to claim 1, is characterized in that described electric field presentation mode is the pulse power or AC power.
3. a capillary column transverse electric chromatographic separation device, includes and provides the pump of transport medium, injector, detector, power supply and one end to be connected injector, and the splitter of other end connection detector is characterized in that described splitter includes:
A capillary, this capillary is as external electrode;
A conductive material, this conductive material is as interior electrode, runs through vertically capillary and in capillary center, in electrode coaxial with external electrode, interior electrode is connected both ends of power with external electrode.
4. a kind of capillary column transverse electric chromatographic separation device according to claim 3, is characterized in that described conductive material is platinum filament, spun gold or filamentary silver.
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Cited By (4)
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| CN104359995A (en) * | 2014-12-04 | 2015-02-18 | 延边大学 | Method for separating bio-macromolecules of flow type stationary phase in column by utilizing electromagnetic field |
| CN104383716A (en) * | 2014-12-04 | 2015-03-04 | 延边大学 | Method for modifying mesoporous material on surface of electrode in transverse electric field capillary column |
| CN105527335A (en) * | 2016-02-02 | 2016-04-27 | 中国科学院电子学研究所 | Photoionization detector |
| CN106512478A (en) * | 2016-11-28 | 2017-03-22 | 延边大学 | Electric field controllable capillary tube size-exclusion chromatography separation method |
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| US8137512B2 (en) * | 2006-09-04 | 2012-03-20 | National Institute Of Advanced Industrial Science And Technology | Process for analyzing sample by capillary electrophoresis method |
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| CN1736538A (en) * | 2005-08-02 | 2006-02-22 | 天津大学 | Method for enhancing dynamic absorption volume of ion exchange absorbent of transverse electric field |
| CN2911691Y (en) * | 2006-06-21 | 2007-06-13 | 戴朝政 | Detention eliminating chromatograhic column |
| US8137512B2 (en) * | 2006-09-04 | 2012-03-20 | National Institute Of Advanced Industrial Science And Technology | Process for analyzing sample by capillary electrophoresis method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104359995A (en) * | 2014-12-04 | 2015-02-18 | 延边大学 | Method for separating bio-macromolecules of flow type stationary phase in column by utilizing electromagnetic field |
| CN104383716A (en) * | 2014-12-04 | 2015-03-04 | 延边大学 | Method for modifying mesoporous material on surface of electrode in transverse electric field capillary column |
| CN104359995B (en) * | 2014-12-04 | 2015-12-30 | 延边大学 | The separation of biopolymer method of flow-type Stationary liquid in the post utilizing electromagnetic field |
| CN105527335A (en) * | 2016-02-02 | 2016-04-27 | 中国科学院电子学研究所 | Photoionization detector |
| CN106512478A (en) * | 2016-11-28 | 2017-03-22 | 延边大学 | Electric field controllable capillary tube size-exclusion chromatography separation method |
| CN106512478B (en) * | 2016-11-28 | 2018-12-28 | 延边大学 | The controllable capillary size exclusion chromatography separation method of electric field |
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