CN108007994A - A kind of method for measuring Capillary Electrophoresis electroosmotic flow - Google Patents

A kind of method for measuring Capillary Electrophoresis electroosmotic flow Download PDF

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CN108007994A
CN108007994A CN201710499539.XA CN201710499539A CN108007994A CN 108007994 A CN108007994 A CN 108007994A CN 201710499539 A CN201710499539 A CN 201710499539A CN 108007994 A CN108007994 A CN 108007994A
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electroosmotic flow
capillary
voltage
sample
sample introduction
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CN108007994B (en
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徐伟
贺木易
张文静
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Beijing Institute of Technology BIT
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    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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    • G01N27/447—Systems using electrophoresis

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Abstract

The present invention relates to analytical chemistry field, discloses a kind of method for measuring Capillary Electrophoresis electroosmotic flow, and this method includes：Using the buffer solution dissolved with label as sample to capillary sample introduction first；Apply first voltage in the capillary both sides, while apply certain separating pressure, detect the first transit time of the label；By the sample to capillary sample introduction again；Apply second voltage, while application and the big separating pressure in the same direction such as first step operation in the capillary both sides, detect the second transit time of the label；Wherein described first voltage is identical with the second voltage amplitude, opposite polarity；According to first transit time and second transit time, the mobility of electroosmotic flow is obtained.The present invention program improves electroosmotic flow measurement accuracy, and the faint electroosmotic flow of coatings capillary pipe quickly can be measured.

Description

A kind of method for measuring Capillary Electrophoresis electroosmotic flow
Technical field
The present invention relates to analytical chemistry field, specifically, is related to a kind of method for measuring Capillary Electrophoresis electroosmotic flow.
Background technology
Capillary Electrophoresis refers to sample under the action of high-voltage dc, with electroosmotic flow (electro-osmotic Flow, EOF) it is driving force, using vitreous silica capillary as split tunnel, each component carries out efficient, fast because of the difference of mobility The separated novel liquid-phase isolation technics of speed.Electroosmotic flow Producing reason is related with the material of vitreous silica capillary, vitreous silica The SiO of capillary material2It can be acted on highly basic, generate silicate, isoelectric point is about 1.5, therefore molten in common separation buffer In liquid (pH is generally higher than 2), the hydrolyzed silicate of inside pipe wall into Si-OH and discharges H+So that vitreous silica capillary tube inner wall It is often negatively charged, due to electrostatic adsorption, a positively charged sheath is formed in the liquid surface close to tube wall, is referred to as inhaled Attached layer.It is the diffusion layer formed by residual ion, the charge layer on this capillary tube inner wall surface and absorption outside adsorption layer Layer just forms electric double layer.Due to the presence of electric double layer, one layer of positive charge layer is formd in the liquid surface close to tube wall, then hair Solution top layer in tubule forms a columnar cation overcoat, and under the action of extra electric field, which will Carry whole solution to move to anode, this phenomenon is known as electroosmotic flow.The Accurate Determining of electroosmotic flow is imitated for evaluation analysis method Fruit has vital meaning, while is also the precondition for calculating some kinetic parameters.
Salomon K. establish a kind of electroosmotic flow approximate model, and electroosmotic flow is sticked by electric field strength, buffer solution in the model Degree, buffer solution dielectric constant, separation temperature etc. influence, since influence factor is complicated, it is difficult to theoretically calculate, can only pass through Measuring.The common methods of measure electroosmotic flow are at present：Assuming that other chromatographic stationary phases of a component and capillary tube inner wall Effect can ignore, then its sum velocity is the sum of for its electroosmotic flow and electrophoretic velocity.Neutral component electrophoretic velocity is zero, that Migration velocity is equal to the speed of electroosmotic flow.Therefore, can be selected when measuring electroosmotic flow under buffer condition used with neutrality Component as label, measure its transit time, and then calculate to obtain electroosmotic flow.
However, such a mode is very difficult for the measure of faint electroosmotic flow, elapsed time is grown, and accuracy of measurement is low.
The content of the invention
In order to overcome above-mentioned technical problem, the present invention provides a kind of method for measuring Capillary Electrophoresis electroosmotic flow, improve Measurement accuracy.
To achieve these goals, the present invention provides a kind of method for measuring Capillary Electrophoresis electroosmotic flow, including：
Using the buffer solution dissolved with label as sample to capillary sample introduction first；
Apply first voltage and separating pressure in the capillary both sides, detect the first transit time of the label；
By the sample to capillary sample introduction again；
Apply second voltage in the capillary both sides, while apply the separating pressure, detect the of the label Two transit times；Wherein described first voltage is identical with the second voltage amplitude, opposite polarity；
According to first transit time and second transit time, the mobility of electroosmotic flow is obtained.
Further, the sample introduction first and the sample introduction again using hydrodynamic injection, vacuum sample introduction, electric power sample introduction or Siphon sample introduction.
Further, the sample introduction first is identical with input mode with the sample injection time of the sample introduction again.
Further, the sample injection time is 1-600 seconds.
Further, the number range of the first voltage and the second voltage is 1-25000V.
Further, the separating pressure setting range is 1-99mbar.
The method of measure Capillary Electrophoresis electroosmotic flow of the present invention, using the buffer solution dissolved with label as sample Apply first voltage and separating pressure to capillary sample introduction first, and in capillary both sides, when detecting the first migration of label Between.Sample is applied into second voltage in capillary both sides, while apply and first step operation phase to capillary sample introduction again afterwards Same separating pressure, detects the second transit time of label.Wherein first voltage is identical with second voltage amplitude, polarity phase Instead.Foundation the first transit time and the second transit time, obtain the mobility of electroosmotic flow.The scheme of the embodiment of the present invention improves electricity The measurement accuracy of seepage flow, accuracy is higher, and suitable for quickly being measured to the faint electroosmotic flow of coatings capillary pipe, compared to biography System method effectively saves the time, improves precision.
Brief description of the drawings
Fig. 1 is the flow chart of the method for measure Capillary Electrophoresis electroosmotic flow of the present invention；
Fig. 2 is the principle schematic of the method for measure Capillary Electrophoresis electroosmotic flow of the present invention；
Fig. 3 is the transit time figure obtained using conventional method；
Fig. 4 is the transit time figure obtained using the method for measure Capillary Electrophoresis electroosmotic flow of the present invention；
Fig. 5 is that result of the method for measure Capillary Electrophoresis electroosmotic flow of the present invention under the conditions of different pressures compares Figure；
Fig. 6 is result ratio of the method for measure Capillary Electrophoresis electroosmotic flow of the present invention under the conditions of different separation voltages To figure；
Fig. 7 is the principle schematic of the method for measure Capillary Electrophoresis electroosmotic flow of the present invention.
Embodiment
Below with reference to the accompanying drawings the embodiment of the present invention is illustrated.Retouched in the attached drawing of the present invention or a kind of embodiment The elements and features that the elements and features stated can be shown in one or more other drawings or embodiments is combined.Should When note that for purposes of clarity, being eliminated in attached drawing and explanation known to unrelated to the invention, those of ordinary skill in the art Component or processing expression and description.
An embodiment of the present invention provides a kind of method for measuring Capillary Electrophoresis electroosmotic flow, as shown in Figure 1, including following step Suddenly：
S101, using the buffer solution dissolved with label as sample to capillary sample introduction first.
Before electroosmotic flow is measured with this method, first have to select suitable neutral marker.Common selection gist is slow Rush the species and pH value of solution, it is ensured that selected label is in electroneutral in buffer system.Using running buffer and one The label for determining concentration is configured to sample.Phosphate buffer solution (the Na that specific usable pH value is 72HPO4/NaH2PO4)。
Hydrodynamic injection pattern is used to inject a certain amount of sample with certain time, 1~99mbar of pressure limit specifically can be with It is 50mbar.Sample injection time is controlled in 1~600s, can be specifically 3 seconds.Input mode can also use vacuum sample introduction, electric power The mode sample introduction such as sample introduction or siphon sample introduction.
S102, in capillary both sides apply first voltage and separating pressure, detects the first transit time of label.
The scope of first voltage is 1-25000V, can be specifically 10000V.
While first voltage is applied, apply separating pressure assistant analysis, 1~99mbar of the pressure limit.Specifically may be used To be 70mbar.
Using the first transit time t of UV detector recording mark thing1.Except UV detector, can also use glimmering Detectors such as photodetector, mass spectrum etc..
S103, by the sample to capillary sample introduction again.
The sampling condition of S103 is identical with S101.
S104, in capillary both sides apply second voltage and the separating pressure, detects the second transit time of label.
Second voltage is identical with first voltage amplitude, opposite polarity.It is auxiliary to apply the separating pressure identical with S102 at the same time Help analysis.Using the second transit time t of the recording mark thing such as detectors such as UV detector, fluorescence detector or mass spectrum2。
S105, foundation the first transit time and the second transit time, obtain the mobility of electroosmotic flow.
The mobility of known electroosmotic flow can be represented by formula (1)：
μeo=veo/E (1)
Wherein, veoIt is electroosmotic flow speed, E is electric field strength, and E can be by V/LtTo represent, wherein LtIt is the overall length of capillary Degree, V are the magnitude of voltage applied.
Assuming that total length LtCapillary effective length be Le, which is the length from sample introduction end to detection window. In above-mentioned S102, while apply voltage and pressure, the first transit time for measuring neutral marker is t1.Then have：
Le=t1(vp1+veo1) (2)
Wherein vp1For the speed of label under pressure, veo1For migration velocity of the label under electroosmotic flow effect.
Apply at the same time in above-mentioned S104, in identical capillary with the medium big pressure of S102 with waiting big reverse electricity Pressure, it is t to measure neutral marker's transit time2.Then have：
Le=t2(vp2+veo2) (3)
Wherein vp2Lower label speed, v are acted on for pressureeo2For migration velocity of the label under electroosmotic flow effect.
Because S102, S104 are applied with opposite polarity but the identical voltage of size, by the current direction of capillary not Together, size is identical, thus the Joule heat produced in two steps inside capillary is identical, then the viscosity of buffer solution also phase Together.When identical pressure is applied on the buffer solution of identical viscosity, the speed that pressure is given is also consistent.It is meanwhile identical slow Rush system, add the big voltage such as conversely, thus the electroosmotic flow produced be wait it is big reverse.Therefore have：
vp1=vp2 (4)
veo1=-veo2 (5)
Simultaneous (2), (3), (4) and (5), obtains：
veo=| t1-t2|Le/(2t1t2) (6)
(6) are brought into formula (1), are obtained：
μeo=veo/ E=veo Lt/ V=| t1-t2|LeLt/(2t1t2V) (7)
Obtain the mobility of electroosmotic flow.
Fig. 2 shows the principle schematic of the method for measure capillary electroosmotic flow provided in an embodiment of the present invention.Specific electricity Swimming condition is：Separation voltage：10kV/-10kV；Separating pressure：70mbar；Detection wavelength：214nm；Sample introduction：50mbar, continues 3 Second；Temperature：25℃；Buffer solution：Phosphate buffer solution (the Na of pH72HPO4/NaH2PO4).Time corresponding to two wave crests Respectively the first transit time and the second transit time.Because two steps are applied with opposite polarity but the identical voltage of size, lead to The current direction difference of capillary is crossed, size is identical, thus the Joule heat produced in two steps inside capillary is identical, then The viscosity of buffer solution is also identical.When identical pressure is applied on the buffer solution of identical viscosity, the speed that pressure is given Unanimously.Meanwhile identical buffer system, add the big voltage such as conversely, thus the electroosmotic flow produced be wait it is big reverse.In two steps The speed and the vector sum of electroosmotic flow speed that the actual migration speed of sample is given for pressure, pressure gives speed phase between two steps Together, electroosmotic flow speed etc. is big reversely, then the difference of two leg speed degree is twice of electroosmotic flow speed.
Fig. 7 also illustrates the principle schematic of the method for measure capillary electroosmotic flow provided in an embodiment of the present invention.Two In secondary sample introduction, the big separating pressure in the same direction such as be applied with, and apply the opposite voltage of size identical polar, generate wait it is reverse greatly Electroosmotic flow.Sample is detected in detection window.
Embodiment 1：
Fig. 3 and Fig. 4 is respectively illustrated under the same conditions, uses the transit time figure and the embodiment of the present invention of conventional method Measure Capillary Electrophoresis electroosmotic flow method transit time figure.Wherein, separation voltage：10kV/-10kV；Separating pressure： 70mbar；Detection wavelength：214nm；Pressure during sample introduction：50mbar, it is for 3 seconds；Temperature：25℃；Buffer solution：The phosphoric acid of pH7 Salt buffer solution.
Conventional method and method provided in an embodiment of the present invention are all using identical sample concentration, operation architecture and into batten Part.Used neutral marker is DMSO, and concentration is 1 percent (volume ratios), and operation architecture is that the phosphate of pH=7 delays Fliud flushing, the overall length 60cm of capillary, effective length 50cm, internal diameter are 75 μm.
Operated in accordance with conventional methods is as follows：With the sample introduction hydrodynamic injection 3s of 50mbar, analyzed, used using the voltage of 10kV UV detector records sample migration time t.Carry out 5 groups to repeat to test, 5 groups of transit time is brought into public affairs are calculated as below respectively Formula (8), (9),
veo=Le/t (8)
μeo=veo/ E=(LeLt)/(tV) (9)
5 groups of result of calculations are averaged, the mobility as electroosmotic flow.
Method concrete operation step provided in an embodiment of the present invention is as follows：
The first step：Hydrodynamic injection pattern is used with 50mbar hydrodynamic injections 3s；
Second step：Electrophoretic analysis is carried out using 10kV voltages, while applies 70mbar pressure assistant analysis, using ultraviolet inspection Survey device record sample migration time t1；
3rd step：Using the sampling condition sample introduction in the first step；
4th step：Electrophoretic analysis is carried out using the voltage of -10kV equal with second step opposite polarity amplitude, is applied at the same time 70mbar pressure assistant analysis, sample migration time t is recorded using UV detector2。
Repeat the above steps 5 times.Multiplicating can reduce random error.
The t that 5 times are measured1、t2Bring above-mentioned formula (7) into, obtain 5 result of calculations, 5 result of calculations are averaged Value, obtains electroendosmotic mobility.
The measurement result of conventional method and method provided in an embodiment of the present invention compares as shown in table 1.
Table 1
It was found from table 1, Fig. 3 and Fig. 4, compared by new out-of-date methods, determine the reliability of new method, new method has higher Accuracy and repeatability.
Embodiment 2：
During electrophoretic analysis, keep other conditions it is constant, only change separating pressure, respectively 50mbar, 60mbar, Analyzed under the conditions of the separating pressure of 70mbar, 80mbar, 90mbar, five groups of parallel realities are done under the conditions of each separating pressure Test.Probe into different separating pressures whether to measure electroosmotic flow efficiency have an impact.Embodiment 2 and 1 uses identical experiment Condition, including identical long capillary tube, capillary inner diameter, operation architecture and sample.
Handled according to the step described in embodiment 1, respectively 50mbar, 60mbar, 70mbar, 80mbar, Analyzed under the separating pressure of 90mbar.Obtained result is as shown in table 2, Fig. 5.
Table 2
From table 2 can with it is concluded that, the change of separating pressure setting value measures electric osmose to the method for the embodiment of the present invention The stability accuracy of stream does not influence substantially, and there is no certain correlation therebetween.From fig. 5, it can be seen that 5 kinds different Under separating pressure setting, the line of obtained electroendosmotic mobility average value is nearly parallel to X-axis, illustrates under 5 kinds of pressure, all may be used With the relatively accurate electroendosmotic mobility calculated under the deposition condition.
, can not when selecting separating pressure although embodiment 2 illustrates that separating pressure has no measurement result influence Any setting, when first having to ensure to add negative voltage, pressure is more than the speed of electroosmotic flow to the speed of sample, sample is arrived Up to detection window.During electrophoretic analysis, the separating pressure added is bigger, and the time measured needed for electroosmotic flow is faster.
Embodiment 3：
During electrophoretic analysis, keep other conditions constant, only change voltage, respectively in 8kV/-8kV, 10kV/- Analyzed under the voltage conditions of 10kV, 12kV/-12kV, 15kV/-15kV, 18kV/-18kV, 20kV/-20kV, each electricity Three groups of parallel laboratory tests are done under the conditions of pressure.Probe into different analysis voltages whether to measure electroosmotic flow efficiency have an impact.Implement Example 3 and embodiment 1 use identical experiment condition, identical long capillary tube, internal diameter, operation architecture and sample.
Experimental result is obtained as shown in table 3, Fig. 6.
Table 3
From table 3 can with it is concluded that, the method measure electric osmose trickling of the change of voltage setting value to the embodiment of the present invention The stability accuracy of degree does not influence substantially, and there is no certain correlation therebetween.From fig. 6, it can be seen that in 5 kinds of voltages Under, the electroendosmotic mobility calculated under the deposition condition that can be relatively accurate, the company for the average mobility that 5 kinds of voltages are set Line is nearly parallel to X-axis, illustrates under different voltage, and this method can compare the electric osmose trickling under the conditions of Accurate Determining is somebody's turn to do Degree.
Embodiment 3 illustrates that the change of analysis voltage does not influence to measure the efficiency of electroosmotic flow.
Embodiment 4：
The method of measure Capillary Electrophoresis electroosmotic flow provided in an embodiment of the present invention is particularly suited for quickly measuring coating hair Faint electroosmotic flow in tubule.
Capillary in embodiment 1 to embodiment 3 is not modified by coating or other inner walls, and capillary wall has Si-OH Base, meets buffer solution ionization, Si-OH dissociates a H ion, makes tube wall negatively charged, close to the buffer solution surface of tube wall Positively charged, after applying voltage and producing electric field, solution can integrally be moved to anode, and here it is the mechanism that electroosmotic flow produces.
Coatings capillary pipe is to carry out coating to capillary tube inner wall, keeps apart the Si-OH of buffer solution and inside pipe wall, so There will be no Si-OH dissociation to lose H+Negatively charged, the solution close to capillary will not be positively charged, will not produce electroosmotic flow. But this is perfect condition, there are faint electroosmotic flow in coated pipe under actual conditions.If using conventional method, the faint electricity is measured It is more than hour to need 1 for seepage flow, and is only needed 10 minutes or so using method minute provided in an embodiment of the present invention.
Experiment condition is as follows：Used coating capillary is the neutral coated pipe of Ao Taike bio tech ltd.Coating Capillary total length 60cm, effective length 50cm, internal diameter are 75 μm, and used neutral marker is DMSO, concentration percent One (volume ratio), operation architecture are the phosphate buffer of pH=7.
The first step：Hydrodynamic injection pattern is used with 50mbar hydrodynamic injections 3s；
Second step：Electrophoretic analysis is carried out using 10kV voltages, while applies 70mbar pressure assistant analysis, using ultraviolet inspection Survey device record sample migration time t1；
3rd step：Using sampling condition sample introduction in the first step；
4th step：Electrophoretic analysis is carried out using the voltage of -10kV equal with second step opposite polarity amplitude, is applied at the same time 70mbar pressure assistant analysis, sample migration time t is recorded using UV detector2。
Repeat the above steps 5 times.
The t that 5 times are measured1、t2Bring above-mentioned formula (7) into, obtain 5 result of calculations, 5 result of calculations are averaged Value, obtains the mobility of electroosmotic flow.Obtain that the results are shown in Table 4.
Table 4
The time for measuring electroosmotic flow in primary coating pipe is about 10 minutes, it is seen that this method can be used for quick measure and apply Faint electroosmotic flow in layer pipe, and the accuracy of this method and repeatability are also proven in embodiment 1.It is of the invention real The method for applying the measure Capillary Electrophoresis electroosmotic flow described in example is particularly suitable for carrying out soon the faint electroosmotic flow of coatings capillary pipe Speed measure, effectively saves the time compared to conventional method, improves measurement accuracy.
Although the present invention and its advantage is described in detail it should be appreciated that without departing from by appended claim Various changes, replacement and conversion can be carried out in the case of the spirit and scope of the present invention limited.Moreover, the model of the application Enclose and be not limited only to the described process of specification, equipment, means, the specific embodiment of method and steps.In the art is common Technical staff will readily appreciate that from the disclosure, can use perform and corresponding reality described herein according to the present invention Apply the essentially identical function of example or obtain process essentially identical with it result, existing and that future is to be developed, equipment, Means, method or step.Therefore, appended claim includes such process, equipment, hand in the range of being directed at them Section, method or step.

Claims (6)

  1. A kind of 1. method for measuring Capillary Electrophoresis electroosmotic flow, it is characterised in that including：
    Using the buffer solution dissolved with label as sample to capillary sample introduction first；
    Apply first voltage and separating pressure in the capillary both sides, detect the first transit time of the label；
    By the sample to capillary sample introduction again；
    Apply second voltage in the capillary both sides, while apply the separating pressure, detect the label second moves Shift time；Wherein described first voltage is identical with the second voltage amplitude, opposite polarity；
    According to first transit time and second transit time, the mobility of electroosmotic flow is obtained.
  2. 2. it is according to claim 1 measure Capillary Electrophoresis electroosmotic flow method, it is characterised in that the sample introduction first and The sample introduction again uses hydrodynamic injection, vacuum sample introduction, electric power sample introduction or siphon sample introduction.
  3. 3. it is according to claim 1 measure Capillary Electrophoresis electroosmotic flow method, it is characterised in that the sample introduction first and The sample injection time of the sample introduction again is identical with input mode.
  4. 4. the method for measure Capillary Electrophoresis electroosmotic flow according to claim 3, it is characterised in that the sample injection time is set It is scheduled in the range of 1-600 seconds.
  5. 5. it is according to claim 1 measure Capillary Electrophoresis electroosmotic flow method, it is characterised in that the first voltage and The number range of the second voltage is 1-25000V.
  6. 6. the method for measure Capillary Electrophoresis electroosmotic flow according to claim 1, it is characterised in that the separating pressure Number range is 1-99mbar.
CN201710499539.XA 2017-06-27 2017-06-27 A method of measurement Capillary Electrophoresis electroosmotic flow Expired - Fee Related CN108007994B (en)

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Publication number Priority date Publication date Assignee Title
CN108663428A (en) * 2018-05-29 2018-10-16 北京理工大学 A method of based on mobility electrophoretic determination matter dimensions
CN111272611A (en) * 2018-12-05 2020-06-12 北京理工大学 Method for determining component data in capillary electrophoresis

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US5624539A (en) * 1995-06-19 1997-04-29 The Penn State Research Foundation Real time monitoring of electroosmotic flow in capillary electrophoresis
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US5441613A (en) * 1993-12-03 1995-08-15 Dionex Corporation Methods and apparatus for real-time monitoring, measurement and control of electroosmotic flow
WO1996022151A1 (en) * 1995-01-18 1996-07-25 Dionex Corporation Methods and apparatus for real-time monitoring, measurement and control of electroosmotic flow
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108663428A (en) * 2018-05-29 2018-10-16 北京理工大学 A method of based on mobility electrophoretic determination matter dimensions
CN111272611A (en) * 2018-12-05 2020-06-12 北京理工大学 Method for determining component data in capillary electrophoresis

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