CN101196464A - Laser double-mode micro-volume sample analyzing method and its device - Google Patents
Laser double-mode micro-volume sample analyzing method and its device Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/171—Systems in which incident light is modified in accordance with the properties of the material investigated with calorimetric detection, e.g. with thermal lens detection
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
- G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/171—Systems in which incident light is modified in accordance with the properties of the material investigated with calorimetric detection, e.g. with thermal lens detection
- G01N2021/1712—Thermal lens, mirage effect
Abstract
The invention discloses a laser dual-mode microvolume sample analyzing method based on laser thermal lens and laser retroreflection interference technique and the used device. Under the wavelength of the used laser, test the signal of the substance with light adsorbing characteristic by the laser thermal lens testing mode to each component through liquid phase separation, and test the signal of the substance without fluorescent and light adsorbing characteristic by the laser retroreflection interference test mode. The invention doesn't need to conduct any sample deriving and collecting processing procedures. Because the required checking sample poor is micro-checking pool, the needed sample volume is very small and can reach nano flow at lowest. The used device in the invention is simple to operate, which has low daily maintaining fee, quick testing speed and accurate result, and can be widely used in the characterization and quantification testing to the need checking system including various different chemical substances.
Description
Technical field
The present invention relates to micro-volume sample analyzing method and equipment therefor, especially a kind of analytical approach and equipment therefor that utilizes laser thermal lens, laser retroeflection interference technique to detect the long-pending sample of microbody simultaneously, belong to optical field, also belong to the analytical chemistry instrument field.
Background technology
The liquid phase separation analytical instrument is a development in recent years alanysis instrument the most rapidly, it comprises high performance liquid chromatography (HPLC), Capillary Electrophoresis (CE), chromatography of ions, instruments such as supercritical fluid chromatography (SFC) and micro-fluidic chip, this quasi-instrument have efficiently, fast, characteristics such as amount of samples and reagent consumption be few.The compartment analysis that can be used for inorganic, organic and biomacromolecule has obtained using widely in scientific research and production field.
Separation efficiency and sensitivity are the important technology indexs of weighing this quasi-instrument, and these indexs had both depended on the character of used chromatographic column or capillary column, also are limited by the performance of detecting device simultaneously.Research and development is applicable to that little pool detector of liquid phase separation analytical instrument has become the popular domain of this quasi-instrument development.Ultraviolet absorption detector is the little pool detector of a most widely used class in this quasi-instrument, make photo-detector owing to adopt diode array or CCD device, can realize the quick scanning of ultraviolet spectrum, in chromatogram or electrophoretic separation, obtain real-time ultraviolet spectrum, thereby realize the mensuration of the qualitative and known substance of principal component not from 190 to 800nm.Because the light path of little pool detector is shorter, makes the sensitivity of ultraviolet absorption detector be difficult to improve, and be difficult at present flourish chip electrophoresis detection.Electrochemical detector also is to analyze in liquid phase separation to use a wider class detecting device in the commercial apparatus, it can avoid the short deficiency of ultraviolet absorption detector light path, in the detection of electroactive component, have highly sensitive, advantage such as the good and range of linearity of selectivity is wide, its limitation is that detected material must have good electrochemical activity, be difficult to when being applied to electrophoresis detection eliminate and separate electric current detecting the interference of electric current, only can carry out behind the post and detect, thereby become needing time of a kind of practical commercialization electrophoresis apparatus at the post detecting device from post.Compare with other detection technique, Mass Spectrometer Method (MS) and the coupling of liquid phase separation analytical instrument can obtain the structural information of separation component, are the objects of numerous apparatus manufactures favors always.HPLC-MS, CE-MS, SFC-MS coupling commercialization instrument are introduced to the market at present, but instrument is complicated and expensive, and its connecting interface and sensitivity still need further to improve and improve.The refractive index detector that variation detects based on the material refractive index is as the detecting device of instruments such as HPLC, and this detecting device versatility is good, but sensitivity is not high.The various detecting devices such as chemiluminescence, nuclear magnetic resonance, kapillary resonance of having seen bibliographical information also all in various degree have a under-sensitive problem.Thereby develop the important topic that highly sensitive and detecting device highly versatile is still the development of liquid phase separation analytical instrument.
In general, laser coherence is good, easily is focused into microbeam, is specially adapted to the mensuration of the long-pending sample of microbody, thereby based on the development of all kinds of laser detectors of laser good characteristic with use the popular domain that has become the research of liquid phase separation analytical instrument detecting device.Laser induced fluorescence detector is a kind of little pool detector that the Gao Bingyi of laser class detecting device medium sensitivity becomes commercialization Capillary Electrophoresis instrument.High 2～3 orders of magnitude of its remolding sensitivity ultraviolet absorption detector, but have only the only a few compound under laser excitation, can produce autofluorescence, most compounds need adopt fluorescence labeling or derive after can analyze.Based on the laser thermal lens detectors measure of low light level absorption detecting is not transmission, reflection or scattered light, but the directly absorption of measuring light, compare with ultraviolet absorption detector, sensitivity can improve 2 more than the order of magnitude, in the detection of non-fluorescence and weakly absorbing material, embody highly sensitive, the advantage of high spatial resolution.The laser Raman spectroscopy detecting device also appears in the newspapers, and is not still the stem reality that this class detecting device faces but detection sensitivity is high.At present, laser class detecting device mostly is mono signal and detects, Shang Weijian detecting device of many signals whiles is used for the report of the instrument of liquid phase separation analysis, and except that laser induced fluorescence detector, other laser light signal detector does not become the detecting device that liquid phase separation is analyzed commercial apparatus as yet.
The analytic target of liquid phase separation analytical instrument (particularly biological sample) contains the complication system of multiple material often.This class sample had both contained biomacromolecules such as protein, nucleic acid, contained micromolecule such as dopamine, amino acid again, and the fluorescence of these molecules and absorption characteristic are different.Accurately measure their kind and content, very great for life science and clinical diagnosis meaning.Yet because the restriction of commercial detector is difficult to obtain simultaneously big molecule and micromolecule and has and do not have the information of fluorescence and extinction characteristic material.Therefore, explore simultaneously many signal detectors that all kinds of materials that have and do not have fluorescence and extinction characteristic are carried out Sensitive Detection and will greatly expand the space that liquid phase separation is analyzed the application of commercial apparatus.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, provide a kind of based on laser-induced fluorescence (LIF), the laser double-mode micro-volume sample analyzing method and the equipment therefor of laser retroeflection interference technique, to the each component after liquid phase separation, under used optical maser wavelength, the laser thermal lens detecting pattern detects the signal with extinction characteristic material, the laser retroeflection interferes the detecting pattern detection not have the signal of fluorescence and extinction characteristic material, and makes its optical configuration and signals collecting pattern reach the light signal that obtains laser thermal lens and laser retroeflection interference simultaneously to the liquid phase separation component.
Realize the technical scheme that the object of the invention adopts: a kind of laser double-mode micro-volume sample analyzing method based on laser-induced fluorescence (LIF), laser retroeflection interference technique may further comprise the steps:
1) will remove one section kapillary of external protection in the capillary electrophoresis apparatus as little detection cell, two ends capillaceous are immersed in respectively under the buffer solution liquid level in buffer solution carrying pond and buffering solution carrying pond, the both positive and negative polarity of DC high-voltage power supply is immersed in respectively under the buffer solution liquid level in buffer solution carrying pond and buffering solution carrying pond by cylindrical platinum electrode respectively, replace one of them buffer solution carrying pond with the sample cell that sample is housed during sample introduction, utilize electricity driving or pressure reduction to carry out the sample introduction operation then, after sample introduction finishes former buffer solution carrying pond is reduced to position originally, open the certain high direct voltage of DC high-voltage power supply output then, make the capillary electrophoresis apparatus energising, under the effect of DC high-voltage power supply, the long-pending sample electrophoresis of different microbodys time degree is different, causing different microbodys to amass sample will be with friction speed to capillary motion, and different chemical substances is little detection cell by the kapillary zone of being stripped from external protection coating successively;
2) after the laser beam that semiconductor laser is penetrated successively passes through the modulation of chopper and polaroid, the laser coupled of sending with the thermal lens detection laser at beam splitter becomes with laser beam, the coupled light beam of gained is focused the center of lens focus in little detection cell after seeing through beam splitter, transmission is crossed the coupled laser bundle of little detection cell through high-pass filter elimination pumping laser bundle, detect by photoelectricity testing part again and obtain laser thermal lens detection original signal, by retroeflection scattered light that laser light scattering produced by beam splitter reflection to vertical direction and produce macroscopic regular interference fringe, obtain the laser retroeflection and interfere and detect original signal through detecting by photoelectricity testing part after the modulation of diaphragm, obtain the detection signal under two kinds of detecting patterns of sensitivity more after interfering two kinds of detecting patterns original signal down to be resolved by phase-locked amplification respectively gained laser thermal lens and the laser retroeflection, the gained detection signal is imported computing machine by data collecting card obtain the real time data figure line, realize microbody is amassed the assay determination of sample by the real time data figure line.
Above-mentioned based on laser thermal lens, the laser double-mode micro-volume sample analyzing method detection device thereof of laser retroeflection interference technique, at least comprise the optical detection modulating part, Capillary Electrophoresis part and signal acquisition process part, semiconductor laser in the optical detection modulating part, chopper, polaroid, beam splitter, beam splitter, condenser lens, little detection cell, high-pass filter and photoelectricity testing part are arranged in order, and the center all is positioned at same horizontal line, the beam splitter vertical direction is provided with the thermal lens detection laser, beam splitter is connected with photoelectricity testing part by diaphragm, and wherein little detection cell is one section kapillary removing external protection in the capillary electrophoresis apparatus; Be provided with lock-in amplifier between computing machine in the signal acquisition process part and the photoelectricity testing part, be provided with lock-in amplifier between computing machine and the photoelectricity testing part, chopper is provided with the chopper controller, and the chopper controller is connected with computing machine with photoelectricity testing part by lock-in amplifier.The chopper controller offers lock-in amplifier with phase-locked reference signal, through phase-locked amplification process, lock-in amplifier provides laser thermal lens signal, laser retroeflection interference signal respectively, gathered by computing machine then, thereby realization is to the assay determination of the long-pending sample of microbody.
Described little detection cell is made by the high material of light transmission rate such as glass, organic glass, quartz etc., and it is shaped as cube or right cylinder, and internal diameter is greater than 75 microns.
Described photoelectricity testing part is optical diode, photomultiplier, photoelectric coupled device or diode array.
Described semiconductor laser is each band laser, and light beam luminous energy is greater than 20 milliwatts.
Described thermal lens detection laser is each band laser, and light beam wavelength is greater than the semiconductor laser beam wavelength.
Distance between the photoelectricity testing part of described laser thermal lens detection signal and the sample cell in the capillary electrophoresis apparatus needs more than or equal to 150 millimeters.
Described condenser lens is a biconvex lens, and its focal length is greater than 9 millimeters and less than 15.4 millimeters, and pinpointed focus is 11 millimeters.
Laser double-mode micro-volume sample analyzing method provided by the present invention combines with liquid phase separation method and treats the micrometer volume sample and carry out separation detection, need not carry out any analyte derivative, enrichment treatment step to sample.Because the testing sample pond is little detection cell, required sample size is few, minimum can be to receiving upgrading.Equipment therefor of the present invention is simple to operate, and the daily servicing expense is low, and test speed is fast, and the result is accurate, can be widely used in including the qualitative and quantitative analysis of the system to be measured of all kinds of different chemical materials.
Description of drawings
The invention will be further described below in conjunction with drawings and Examples.
Fig. 1 is the optical texture synoptic diagram of laser double-mode micro-volume sample analyzing method equipment therefor of the present invention.
Fig. 2 is that the used electrophoresis apparatus system architecture of laser double-mode micro-volume sample analyzing method synoptic diagram is interfered in laser thermal lens, laser retroeflection.
Fig. 3 is the signals collecting part-structure synoptic diagram of laser double-mode micro-volume sample analyzing method equipment therefor of the present invention.
Among the figure: 1. semiconductor laser, 2. chopper, 3. polaroid, 4. beam splitter, 5. thermal lens detection laser, 6. beam splitter, 7. diaphragm, 8. optical diode, 9. condenser lens, 10. little detection cell (peeling off the kapillary of external protection coating), 11. high-pass filters, 12. optical diode, 13. kapillaries, 14. DC high-voltage power supply, 15. buffer solution carrying pond, 16. sample cells, 17. buffer solution carrying pond, 18. chopper controllers, 19. lock-in amplifier, 20. lock-in amplifiers, 21. computing machines.
Embodiment
The present invention is based on laser thermal lens, the optical texture of the laser double-mode micro-volume sample analyzing method detection device thereof of laser retroeflection interference technique as shown in Figure 1, at least comprise the optical detection modulating part, Capillary Electrophoresis part and signal acquisition process part, semiconductor laser 1 in the wherein optical detection modulating part, chopper 2, polaroid 3, beam splitter 4, beam splitter 6, condenser lens 9, little detection cell 10, high-pass filter 11 and photoelectricity testing part 12 are arranged in order, and the center all is positioned at same horizontal line, beam splitter 4 vertical direction are provided with thermal lens detection laser 5, beam splitter 6 is connected with photoelectricity testing part 8 by diaphragm 7, and wherein little detection cell 10 is a kapillary of removing one section external protection in the capillary electrophoresis apparatus; Be provided with lock-in amplifier 19 between computing machine 21 in the signal acquisition process part and the photoelectricity testing part 8, be provided with lock-in amplifier 20 between computing machine 21 and the photoelectricity testing part 12, chopper 2 is provided with chopper controller 18, and chopper controller 18 is connected with computing machine 21 with photoelectricity testing part 12 by lock-in amplifier 19.Wherein semiconductor laser 1 is for can select each band laser for use, and light beam luminous energy is greater than 20 milliwatts.The frequency of operation of chopper 2 is 5Hz to 1000Hz; The splitting ratio of beam splitter 4 and beam splitter is 5: 5, and its beam splitting wave band corresponds respectively to the wave band of semiconductor laser and thermal lens detection laser; The thermal lens detection laser can be selected each band laser for use, and its light beam wavelength is greater than the semiconductor laser beam wavelength; Photoelectricity testing part 8 and photoelectricity testing part 12 are optical diode, photomultiplier, photoelectric coupled device, diode array; Condenser lens 9 is a biconvex lens, and less than 15.4 millimeters, pinpointed focus is 11 millimeters to its focal length greater than 9 millimeters; 1 little detection cell 0 is made for the high material of light transmission rate such as glass, organic glass, quartz etc., and it is shaped as cube or right cylinder, and its internal diameter is greater than 75 microns; The selection of high-pass filter 11 wave bands should be selected according to the wave band situation of semiconductor laser and thermal lens detection laser, and it is higher than the wave band of semiconductor laser by wave band; Polaroid 3 and diaphragm 7 do not have is strict with, and the product of each technical parameter all can meet the demands; The photoelectricity testing part of laser thermal lens detection signal and the distance between the sample cell are greater than or equal 150 millimeters.
After semiconductor laser 1 emitted laser beam passes through the modulation of chopper 2 and polaroid 3, be coupled into laser beam with thermal lens detection laser 5 emitted laser beams at beam splitter 4 places, the gained coupled light beam is focused the center that lens 9 focus on little detection cell 10 after seeing through beam splitter 6; The coupled laser bundle that sample cell is crossed in transmission is through high-pass filter 11 elimination pumping laser bundles, and then detected by photoelectricity testing part 12 and to obtain laser thermal lens detecting pattern original signal; The sample cell place obtains the laser retroeflection and interferes the detecting pattern original signal through being detected by photoelectricity testing part 8 after the modulation of diaphragm 7 because retroeflection scattered light that scattering produced reflexes to vertical direction and produces macroscopic regular interference fringe through beam splitter 6.
The used capillary electrophoresis apparatus of laser double-mode micro-volume sample analyzing method that the present invention is based on laser thermal lens, laser retroeflection interference technique constitutes as shown in Figure 2.The two ends of kapillary 13 are immersed in respectively under the buffer solution liquid level in buffer solution carrying pond 15 and buffering solution carrying pond 17.The both positive and negative polarity of DC high-voltage power supply 14 links to each other with cylindrical platinum electrode respectively, and two electrodes are immersed in respectively under the buffer solution liquid level in buffer solution carrying pond.With one of them buffer solution carrying pond 15 of sample cell 16 replacements that microbody amasss sample is housed, utilize electricity driving or pressure reduction to carry out sample introduction then and operate during sample introduction.External protection capillaceous is removed (adopt naked light is ablated or sharp sword strikes off mode all can) in position suitable on kapillary 13; one section that kapillary is peeled off external protection coating as little detection cell 10, thereby realizes the real-time detection of detection cell detecting device to the Capillary Electrophoresis dynamic process that decline of laser multimode.Wherein kapillary 13 be various outer diameter parameters kapillary all can, internal diameter is more than or equal to 75 microns; High-voltage DC power supply 14, its maximum output voltage should be greater than 20 kilovolts; Buffer solution carrying pond 15, buffer solution carrying pond 17, sample cell 16, its material need be selected insulator for use.
The present embodiment signal acquisition process as shown in Figure 3.Be provided with lock-in amplifier 19 between computing machine 21 in the signal acquisition process part and the photoelectricity testing part 8, be provided with lock-in amplifier 20 between computing machine 21 and the photoelectricity testing part 12, and chopper controller 18 is connected with computing machine 21 with photoelectricity testing part 12 by lock-in amplifier 19.Carry out in the electrophoresis process interfering at the described electrophoresis apparatus of Fig. 2 and detect original signal and laser thermal lens detects original signal from the laser retroeflection that photoelectricity testing part 8, photoelectricity testing part 12 obtain current system.Then two kinds of signals are imported lock-in amplifier 19, lock-in amplifier 20 respectively, in conjunction with the reference signal that imports from chopper controller 18, obtain the lower laser thermal lens of ground unrest by phase-locked processing and amplifying and detect signal to be collected, laser retroeflection interference detection signal to be collected.By personal computer 21 subsidiary digital collection cards three kinds of above-mentioned signals to be collected are gathered, obtained data figure line, thereby realize microbody is amassed the assay determination of sample according to the real time data figure line corresponding to real-time change.
Concrete testing process:
At first kapillary 13 is washed with 0.1mol/LNaOH solution, redistilled water and buffer solution successively, carry in the pond 17 and be full of buffer solution at buffer solution carrying pond 15, buffer solution, and the two ends of kapillary 13 are immersed in respectively under the buffer solution liquid level in buffer solution carrying pond 15 and buffering solution carrying pond 17.The both positive and negative polarity of DC high-voltage power supply 14 links to each other with cylindrical platinum electrode respectively, and two electrodes are immersed in respectively under the buffer solution liquid level in buffer solution carrying pond 15 and buffering solution carrying pond 17.
In beginning electrophoresis process precontract half an hour, that semiconductor laser 1, chopper 2, thermal lens detection laser 5, photoelectricity testing part 8, photoelectricity testing part 12, lock-in amplifier 19, lock-in amplifier 20, chopper controller 18, computing machine 21 energisings is relatively stable until the gained signal.
With being housed, the long-pending sample sample cell 16 of microbody replaces one of them buffer solution carrying pond 15 during sample introduction, utilize electricity driving or pressure reduction to carry out sample introduction sample introduction operation certain hour then, after sample introduction finishes former buffer solution carrying pond 15 is reduced to position originally, opens the certain high direct voltage of DC high-voltage power supply 14 outputs then.After the energising, under the effect of DC high-voltage power supply 14, different chemical substance electrophoresis time degree is different, causes different chemical substances to move to kapillary 13 ends with friction speed.Through certain hour, different chemical substances is successively by being stripped from the kapillary zone 10 of external protection coating, utilize laser double-mode micro-volume sample analyzing method detection device thereof provided by the present invention to detect then, from photoelectricity testing part 8, photoelectricity testing part 12 places obtain respectively interfering the detection original signal corresponding to the laser retroeflection of variant chemical substance, laser thermal lens detects original signal (being all the voltage data of hundreds of millivolts of sizes), pass through phase-locked processing and amplifying process then, be respectively the data collecting card collection and import computing machine 21, thereby realized detection the real-time electrophoretic separation process of system to be measured.The appearance of each peak correspondence is the foundation of variant chemical substance observational measurement in proper order in the gained figure line, and the peak height of each peak correspondence or peak area are the foundations of variant chemical substance quantitative measurment.
Following discloses are specifically implemented test result for one group:
Semiconductor laser 1 laser wavelength is 532nm.Beam splitter 4 is selected wave band 532nm for use, and splitting ratio is 5: 5 a beam splitter.Thermal lens detection laser 5 is selected 632.8nm for use, 2 milliwatt helium-neon lasers, and beam splitter 6 is selected wave band 632.8nm for use, and splitting ratio is 5: 5 a beam splitter.Photoelectricity testing part 8 and photoelectricity testing part 12 are selected optical diode for use, and condenser lens 9 is selected the biconvex condenser lens for use, and its focal length is 11 millimeters.High-pass filter 11 is selected cutoff wavelength 600nm for use, the high-pass filter of cut-off width 20nm.40 centimetres of kapillary 13 length, 100 microns of internal diameters; Removing external protection capillaceous zone is that little detection cell 10 is from 5 centimetres of kapillary 13 tail ends.With the 12.5mol/L borax soln as buffer solution; sample introduction voltage and separation voltage are 9KV; sample introduction 8 seconds; about 15 minutes of detection time; at the little detection cell of the kapillary of removing external protection 10 places; isolate DOPA ammonia, amaranth (without any example enrichment or derivation process) successively, adopt laser double-mode micro-volume sample analyzing method detection device thereof of interfering provided by the invention to obtain two width of cloth figure line of corresponding laser thermal lens detection signal, laser retroeflection interference detection signal respectively based on laser thermal lens, laser retroeflection.According to the separated order of DOPA ammonia, amaranth and the size of peak area, detect the concentration of these chemical substances in the testing sample, the detection limit of these two kinds of chemical substances: DOPA ammonia is 5.0 * 10
-6Mol/L, amaranth is 2 * 10
-9Mol/L.
Claims (10)
1. laser double-mode micro-volume sample analyzing method, it is characterized in that may further comprise the steps: 1) will remove one section kapillary (13) of external protection in the capillary electrophoresis apparatus as little detection cell (10), the two ends of kapillary (13) are immersed in respectively under the buffer solution liquid level in buffer solution carrying pond (15) and buffering solution carrying pond (17), the both positive and negative polarity of DC high-voltage power supply (14) is immersed in respectively under the buffer solution liquid level in buffer solution carrying pond (15) and buffering solution carrying pond (17) by cylindrical platinum electrode respectively, replace one of them buffer solution carrying pond with the sample cell (16) that sample is housed during sample introduction, utilize electricity driving or pressure reduction to carry out the sample introduction operation then, after sample introduction finishes former buffer solution carrying pond is reduced to position originally, open the certain high direct voltage of DC high-voltage power supply (14) output then, make the capillary electrophoresis apparatus energising, under the effect of DC high-voltage power supply, the long-pending sample electrophoresis of different microbodys time degree is different, cause the long-pending sample of different microbodys to move to kapillary (13) with friction speed, making chemical substances different in the sample is little detection cell (10) by the kapillary zone that is removed external protection coating successively;
2) after the laser beam that semiconductor laser (1) is penetrated successively passes through the modulation of chopper (2) and polaroid (3), the laser coupled of sending with thermal lens detection laser (5) at beam splitter (4) becomes with laser beam, the coupled light beam of gained is focused the center that lens (9) focus on little detection cell (10) after seeing through beam splitter (6), transmission is crossed the coupled laser bundle of little detection cell (10) through high-pass filter (11) elimination laser beam, detect by photoelectricity testing part (12) again and obtain laser thermal lens detection original signal, reflex to vertical direction and produce macroscopic regular interference fringe by beam splitter (6) by the retroeflection scattered light that laser light scattering produced, obtain the laser retroeflection and interfere to detect original signal through detecting by photoelectricity testing part (8) after the modulation of diaphragm (7), obtain the detection signal under two kinds of detecting patterns of sensitivity more after interfering two kinds of detecting patterns original signal down to be resolved by phase-locked amplification respectively gained laser thermal lens and the laser retroeflection, the gained detection signal is imported computing machine by data collecting card obtain the real time data figure line, realize microbody is amassed the assay determination of sample by the real time data figure line.
2. according to the described laser double-mode micro-volume sample analyzing method of claim, it is characterized in that: used photoelectricity testing part (8) and photoelectricity testing part (12) are optical diode, photomultiplier, photoelectric coupled device or diode array.
3. the described laser double-mode micro-volume sample analyzing method equipment therefor of claim 1, at least comprise the optical detection modulating part, Capillary Electrophoresis part and signal acquisition process part, it is characterized in that: the semiconductor laser in the optical detection modulating part (1), chopper (2), polaroid (3), beam splitter (4), beam splitter (6), condenser lens (9), little detection cell (10), high-pass filter (11) and photoelectricity testing part (12) are arranged in order, and the center all is positioned at same horizontal line, beam splitter (4) vertical direction is provided with thermal lens detection laser (5), beam splitter (6) is connected with photoelectricity testing part (8) by diaphragm (7), and wherein little detection cell (10) is a section that kapillary in the capillary electrophoresis apparatus (13) is removed external protection; Be provided with lock-in amplifier (19) between computing machine (21) in the signal acquisition process part and the photoelectricity testing part (8), be provided with lock-in amplifier (20) between computing machine (21) and the photoelectricity testing part (12), chopper (2) is provided with chopper controller (18), and chopper controller (18) is connected with computing machine (21) with photoelectricity testing part (12) by lock-in amplifier (19).
4. according to the described laser double-mode micro-volume sample analyzing method equipment therefor of claim 3, it is characterized in that: little detection cell (10) is made by the high material of light transmission rate, and it is shaped as cube or right cylinder, and internal diameter is greater than 75 microns.
5. according to claim 3 or 4 described laser double-mode micro-volume sample analyzing method equipment therefors, it is characterized in that: little detection cell (10) is made for glass, organic glass, quartz.
6. according to the described laser double-mode micro-volume sample analyzing method equipment therefor of claim 3, it is characterized in that: photoelectricity testing part is optical diode, photomultiplier, photoelectric coupled device or diode array.
7. according to the described laser double-mode micro-volume sample analyzing method equipment therefor of claim 3, it is characterized in that: semiconductor laser (1) is each band laser, and light beam luminous energy is greater than 20 milliwatts.
8. according to the described laser double-mode micro-volume sample analyzing method equipment therefor of claim 3, it is characterized in that: thermal lens detection laser (5) is each band laser, and light beam wavelength is greater than the semiconductor laser beam wavelength.
9. according to the described laser double-mode micro-volume sample analyzing method equipment therefor of claim 3, it is characterized in that: the distance between the sample cell (16) in photoelectricity testing part (12) and the capillary electrophoresis apparatus is more than or equal to 150 millimeters.
10. according to the described laser double-mode micro-volume sample analyzing method equipment therefor of claim 3, it is characterized in that: condenser lens (9) is a biconvex lens, and its focal length is greater than 9 millimeters and less than 15.4 millimeters, and pinpointed focus is 11 millimeters.
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