CN100549677C - Oblique capillary electrophoretic chemical luminescent is dual at sleeved piled inspecting interface - Google Patents
Oblique capillary electrophoretic chemical luminescent is dual at sleeved piled inspecting interface Download PDFInfo
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- CN100549677C CN100549677C CNB2007100091477A CN200710009147A CN100549677C CN 100549677 C CN100549677 C CN 100549677C CN B2007100091477 A CNB2007100091477 A CN B2007100091477A CN 200710009147 A CN200710009147 A CN 200710009147A CN 100549677 C CN100549677 C CN 100549677C
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Abstract
It is dual at sleeved piled inspecting interface to the invention provides a kind of oblique capillary electrophoretic chemical luminescent, and detection interface of the present invention partly is made up of separation capillary, reaction kapillary I, reaction kapillary II, air exhaust pool, chemiluminescence detection window and waste liquid outlet etc.The Interface design of tilting has farthest prevented the premixed of reactant liquor I and reactant liquor II, guarantee that it participates in chemiluminescence reaction with optium concentration, thereby improve relative luminous intensity effectively, improve detection sensitivity, reduce the broadening of bands of a spectrum, improve separation efficiency.The present invention in the end first order reaction end capillaceous has increased an air exhaust pool, solves well and produces gas bubble blockage problem capillaceous in the chemiluminescence reaction process.The present invention helps promoting the development and the application of Capillary Electrophoresis and chemiluminescence coupling technology for capillary electrophoresis chemiluminescence provides a desirable detection interface.
Description
Technical field
The invention belongs to the analytical chemistry field, it is dual at sleeved piled inspecting interface more specifically to relate to a kind of oblique capillary electrophoretic chemical luminescent.
Background technology
The capillary electrophoresis separation technology comes across the seventies in last century the earliest, because of its separation efficiency height, velocity of separation are fast, sample consumption less, tripping device simply reaches advantages such as running cost is low, makes it become a kind of strong separate analytical technique.Yet, because the restriction of capillary size makes sample volume minimum, to obtain higher sensitivity like this, just the development to capillary tube electrophoresis detector has proposed challenge.At present, use more detecting device in the Capillary Electrophoresis ultraviolet, fluorescence, mass spectrum, galvanochemistry and nuclear magnetic-resonance detector etc. are arranged.The sensitivity that these detecting devices have is low, and as UV-detector, the instrument costliness or the practical ranges that have are narrow, as laser induced fluorescence detector.Mass spectrum, galvanochemistry or nuclear magnetic resonance etc. and kapillary coupling technique, detection sensitivity is than the high 1-5 of a ultraviolet detection order of magnitude, but these equipment also need the corollary apparatus of other more complicated except costing an arm and a leg, and Operation and maintenance are all inconvenient.Chemiluminescence detection The Application of Technology highly sensitive, easy and simple to handle makes the range of application of Capillary Electrophoresis obtain [Y.M.Liu, etc.J.Chromatogr.A, 2002,959 of widening to a certain degree; C.Kuyper, etc.Trends Anal.Chem., 2001,414 (5): 1; X.J.Huang, etc.Anal.Chim.Acta, 2000,414:1].
Be that (CE is the abbreviation of capillary electrophoresis for the CE of detected object with the chemiluminescence, the Chinese meaning is " Capillary Electrophoresis ") detecting device do not need excitation source, the photon that only needs a photomultiplier or other photoelectric conversion devices to receive to be sent in the chemical reaction process is also measured comparison to light intensity, just can reach the purpose of substance quantitative analysis.But, (CL is the abbreviation of Chemiluminescence in order to cause the CL reaction, the Chinese meaning for " chemiluminescence), usually all need be to one or both reaction reagents of the online introducing of this detection architecture, this just needs suitable chemiluminescence Detection in Capillary Electrophoresis interface of design.In order to obtain separating effect and higher sensitivity preferably, the factor of overriding concern is exactly the mixed mode and the detection volume of analyte and chemiluminescent reactant.During this external design in order to obtain a stable system, also must consider the pH value when being used for CE separates and be used to keep the compatibility of used chemical illuminating reagent chemiluminescence pH value and the stability of part chemical illuminating reagent (as H
2O
2).Design a well behaved CE-CL connecting interface, effectively carry out, overcome separation component in the diffusion that detects the position with to obtain higher sensitivity all extremely important for what guarantee chemiluminescence reaction.According to the introducing mode of reagent behind post, the chemiluminescence Detection in Capillary Electrophoresis interface arrangement has following several modes: post rear sleeve formula [HuangX-J, etc.Anal Chim Acta, 2000,414,1～14; Zhu A, etc.Kexue Tongbao, 1990,35,672], at column sleeve tubular type [Hara T, etc.Anal Sci, 1991,7:261; Dadoo R, Seto AG, etc.High ResolutChromatogr, 1992,15:133.] and styletable liquid pool formula [Dadoo R, etc.Anal Chem, 1994,66:303～306; Hashimoto M, etc.J Chromatogr A, 1999,832:191～202.] etc.
Reagent was introduced interface after early stage chemiluminescence Detection in Capillary Electrophoresis interface had only a post.Yet in order to cause the CL reaction, many chemical luminous systems need two or more reaction reagents of online introducing.Early stage way is earlier these several reagent to be mixed before introducing luminous pond, and then is introduced in the interface inflow reaction kapillary by unique luminescence reagent.But this disposal route produces some problems sometimes, mix the decomposition that can cause hydrogen peroxide such as hydrogen peroxide in advance with NaOH, the concentration of hydrogen peroxide that arrives reaction zone is reduced, thereby cause the reduction of luminescence reagent luminescence efficiency, and the bubble that hydrogen peroxide decomposes produces also often stops up kapillary, make the kapillary electric current produce drift, thus the reappearance that influence detects.Moreover, the reaction reagent in some luminescence system be can not carry out premixed.Chemiluminescence such as the acridinium ester class normally will be carried out acidifying with the testing sample solution that contains acridinium ester earlier, and then go to cause its chemiluminescence with alkali, the souring soln of wherein using can not mix in advance with alkali lye, otherwise must have a kind of function to completely lose.
Goal of the invention
The objective of the invention is to provide a kind of oblique capillary electrophoretic chemical luminescent dual at sleeved piled inspecting interface, the Interface design of the tilting of this interface effectively improves relative luminous intensity, improve detection sensitivity, reduce the broadening of bands of a spectrum, improve separation efficiency and improved the reappearance that detects, help promoting the development and the application of Capillary Electrophoresis and chemiluminescence coupling technology.
Oblique capillary electrophoretic chemical luminescent of the present invention is dual at sleeved piled inspecting interface, comprise separation capillary, reaction kapillary I, reaction kapillary II, it is characterized in that: II is coaxial with reaction kapillary I, reaction kapillary for described separation capillary, and its axis becomes the angle of inclination with surface level; Described separation capillary is formed the double gradient sleeve pipe with reaction kapillary I, reaction kapillary II, promptly reacts kapillary I and is enclosed within the overlapping of the terminal of separation capillary and some, and reaction kapillary II is enclosed within the terminal of reaction kapillary I and some is overlapping; Described reaction kapillary I is provided with reactant liquor I inlet; Described reaction kapillary II is provided with reactant liquor II inlet; Be provided with the luminous detection window under the described reaction kapillary II; Described reaction kapillary II end is provided with air exhaust pool; Described air exhaust pool middle and lower part is provided with separation capillary electrophoresis earth terminal, and described air exhaust pool also is provided with waste liquid outlet, and described reaction kapillary II end is located at the air exhaust pool middle and lower part and is lower than waste liquid outlet.
Remarkable advantage of the present invention is:
(1) Interface design of tilting of the present invention has farthest prevented reactant liquor I and reactant liquor II mixing before chemiluminescence reaction takes place, guarantee that it participates in chemiluminescence reaction with optium concentration, thereby improve relative luminous intensity effectively, improve detection sensitivity, reduce the broadening of bands of a spectrum, improve separation efficiency.
(2) many chemiluminescence reactions need be added hydrogen peroxide as oxygenant, and the decomposition of hydrogen peroxide under alkali condition can produce bubble, and stop up kapillary or make the kapillary electric current produce fluctuation, thus the reappearance that influence detects.The present invention in the end first order reaction end capillaceous has increased an air exhaust pool, has solved the problems referred to above well.
(3) chemiluminescence detector has many advantages than other detecting device of Capillary Electrophoresis, and such as highly sensitive, cost is lower etc., but owing to many technical elements problems, is not well used at present.The present invention helps promoting the development and the application of Capillary Electrophoresis and chemiluminescence coupling technology for capillary electrophoresis chemiluminescence provides a desirable detection interface.
Description of drawings
Fig. 1 is for using the chemiluminescence Detection in Capillary Electrophoresis Instrument structure synoptic diagram that the present invention detects interface, wherein: 1 high-voltage power supply; 2 Capillary Electrophoresis damping fluid liquid storage tanks; 3 separation capillary; 4 oblique capillary electrophoretic chemical luminescents are dual at sleeved piled inspecting interface; 5 reactant liquor I injection pumps; 6 reactant liquor II injection pumps; 7 waste liquid pools; 8 photomultipliers; 9 faint light detecting devices; 10 computing machines.
Fig. 2 is that oblique capillary electrophoretic chemical luminescent is dual at sleeved piled inspecting interface device design diagram, wherein: 11 organic glass pedestals; 12 reactant liquor I inlet; 13 reactant liquor II inlet; 14 reaction kapillary I; 15 reaction kapillary II; 16 air exhaust pool; 17 platinum electrodes (electrophoresis earth terminal); 18 waste liquid outlets; 19 luminous detection windows.
Fig. 3 detects acridinium ester [4-(2-hydroxyl succinoamino carbonyl ethyl) phenyl-10-methylacridine-9-carboxylate fluoro sulfonate] working curve (AE) for using apparatus of the present invention.
Deposition condition: the separation capillary total length is 600mm, and internal diameter is 75 μ m; Running buffer is 10mmol/L, the phosphate buffer of pH=8.0; Working voltage: 20kV; The photomultiplier high pressure is 1150V; Sample introduction: 20KV electrokinetic injection 15s.
Fig. 4 detects the observed bands of a spectrum of the AE effect that narrows down for apparatus of the present invention
(1) deposition condition: the separation capillary total length is 600mm, and internal diameter is 75 μ m; Running buffer is 10mmol/L, the phosphate buffer of pH=8.0; Working voltage: 20kV; The photomultiplier high pressure is 650V; Sample introduction: 20KV electrokinetic injection 15s.
(2) deposition condition: the separation capillary total length is 750mm, and effective length 600mm, internal diameter are 75 μ m; Running buffer is 10mmol/L, the phosphate buffer of pH=8.0; Working voltage: 20kV; Sample introduction: 20KV electrokinetic injection 15s; The ultraviolet detection wavelength is 265nm.
Embodiment
Oblique capillary electrophoretic chemical luminescent of the present invention is dual at sleeved piled inspecting interface, and its angle of inclination is 15～75 degree; Air exhaust pool is a cylindrical pond, and diameter is more than or equal to three times of the reaction capillary diameter; Reactant liquor I and reactant liquor II are provided through reaction kapillary I and reaction kapillary II by two flow injection pumps respectively; The luminous detection window is 1～10mm along the width of kapillary direction.
Use this device according to the chemiluminescence Detection in Capillary Electrophoresis method.
Embodiment
Application apparatus of the present invention detection acridinium ester [4-(2-hydroxyl succinoamino carbonyl ethyl) phenyl-10-methylacridine-9-carboxylate fluoro sulfonate:
1) deposition condition: the separation capillary total length is 600mm, and internal diameter is 75 μ m; Running buffer is 10mmol/L, the phosphate buffer of pH=8.0; Reactant liquor I is the 0.02mol/L salpeter solution; Reactant liquor II is the NaOH of 0.5mol/L; The flow velocity of two reactant liquor syringe pumps is 0.25 μ l/s; Faint light detecting device photomultiplier transit tube voltage is 1150V; New kapillary washes 20min activation post surface successively with NaOH and the water of HCl, the 1mol/L of 1mol/L, and NaOH and water with 0.1mol/L before starting working every day wash 10min respectively, wash 5min with running buffer again.Working voltage: 20kV.Sample introduction: electrokinetic injection 15s.
2) sample detection: with 1) to prepare a series of concentration be 1.0 * 10 for described electrophoretic buffer
-11～1.0 * 10
-5The AE standard solution of mol/L, by 1) described deposition condition of step carries out capillary electrophoresis separation and detects, and the record luminous signal is a horizontal ordinate with the logarithm of volumetric molar concentration (C) value of AE, logarithm with relative luminous intensity value (I) is an ordinate, draws the working curve (Fig. 3) that detects AE.The linear equation of apparatus of the present invention detection AE is logI=0.4317logC+6.7757 as shown in Figure 3, (R
2=0.9971), detection is limited to 2.85 * 10
-12Mol/L.
3) bands of a spectrum of the interface effect that narrows down
The spectral bandwidth of Capillary Electrophoresis (or half-peak breadth) is one of important parameter of reflection capillary electrophoresis separation efficient, and when other parameter constant, the narrow more then kapillary of bands of a spectrum is strong more to the separating power of this material.Chemiluminescence Detection in Capillary Electrophoresis is because introduced an interface arrangement; usually can cause the bands of a spectrum broadening of detected object, the peak width that the broadening effect of this bands of a spectrum usually can be by its bands of a spectrum relatively and the same terms peak width during time with ultraviolet detection is judged intuitively.Fig. 4 (1) detects 1.0 * 10 with chemiluminescence Detection in Capillary Electrophoresis device of the present invention
-4The spectrogram that mol/LAE obtains, Fig. 4 (2) is the spectrogram that obtains with Capillary Electrophoresis ultraviolet detection same concentrations AE under identical deposition condition, as we can clearly see from the figure, the designed detection interface of the present invention does not only cause the bands of a spectrum broadening of measured matter, but also observed the phenomenon that narrows down of bands of a spectrum to a certain degree, and this mainly is relevant with the quick luminescence-producing reaction of AE, this phenomenon also has report [M.A.Ruberto in other document, etc., Anal.Chem.64 (1992) 2758.]
Claims (5)
1. an oblique capillary electrophoretic chemical luminescent is dual at sleeved piled inspecting interface, comprise separation capillary, reaction kapillary I, reaction kapillary II, it is characterized in that: II is coaxial with reaction kapillary I, reaction kapillary for described separation capillary, and its axis becomes the angle of inclination with surface level; Described separation capillary is formed the double gradient sleeve pipe with reaction kapillary I, reaction kapillary II, promptly reacts kapillary I and is enclosed within the overlapping of the terminal of separation capillary and some, and reaction kapillary II is enclosed within the terminal of reaction kapillary I and some is overlapping; Described reaction kapillary I is provided with reactant liquor I inlet; Described reaction kapillary II is provided with reactant liquor II inlet; Be provided with the luminous detection window under the described reaction kapillary II; Described reaction kapillary II end is provided with air exhaust pool; Described air exhaust pool middle and lower part is provided with separation capillary electrophoresis earth terminal, and described air exhaust pool also is provided with waste liquid outlet, and described reaction kapillary II end is located at the air exhaust pool middle and lower part and is lower than waste liquid outlet.
2. oblique capillary electrophoretic chemical luminescent according to claim 1 is dual at sleeved piled inspecting interface, it is characterized in that: described angle of inclination is 15～75 degree.
3. oblique capillary electrophoretic chemical luminescent according to claim 1 is dual at sleeved piled inspecting interface, it is characterized in that: described air exhaust pool is a cylindrical pond, and diameter is more than or equal to three times of the reaction capillary diameter.
4. oblique capillary electrophoretic chemical luminescent according to claim 1 is dual at sleeved piled inspecting interface, it is characterized in that: described reactant liquor I and reactant liquor II are provided through reaction kapillary I and reaction kapillary II by two flow injection pumps respectively.
5. oblique capillary electrophoretic chemical luminescent according to claim 1 is dual at sleeved piled inspecting interface, it is characterized in that: described luminous detection window is 1～10mm along the width of kapillary direction.
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| CN102661948B (en) * | 2012-05-28 | 2014-04-09 | 福州大学 | Capillary electrophoresis-chemiluminescence detection interface device and preparation method thereof |
| CN107782714A (en) * | 2016-08-24 | 2018-03-09 | 中国科学院光电研究院 | A kind of laser pulse width includes the Laser induced plasma spectroscopy analytical equipment of both psec and nanosecond specification |
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| 毛细管电泳化学发光在线检测. 张颖.分析测试学报，第17卷第1期. 1998 |
| 毛细管电泳化学发光在线检测. 张颖.分析测试学报，第17卷第1期. 1998 * |
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