CN108007994B - A method of measurement Capillary Electrophoresis electroosmotic flow - Google Patents
A method of measurement Capillary Electrophoresis electroosmotic flow Download PDFInfo
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Abstract
The present invention relates to analytical chemistry field, a kind of method for measuring Capillary Electrophoresis electroosmotic flow is disclosed, this method comprises: using the buffer dissolved with marker as sample to capillary sample introduction for the first time；Apply first voltage in the capillary two sides, while applying certain separating pressure, detects the first transit time of the marker；By the sample to capillary sample introduction again；Apply second voltage, while the big separating pressure in the same direction such as application and first step operation in the capillary two sides, detects the second transit time of the marker；Wherein the first voltage is identical as the second voltage amplitude, and polarity is opposite；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 can quickly be measured to the faint electroosmotic flow of coatings capillary pipe.
Description
Technical field
The present invention relates to analytical chemistry fields, specifically, being related to a kind of method for measuring Capillary Electrophoresis electroosmotic flow.
Background technique
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 novel liquid-phase isolation technics of speed separation.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 at Si-OH and releases 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 outside adsorption layer, the charge layer on this capillary tube inner wall surface and absorption
Layer just constitutes 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 surface layer in tubule forms a columnar cationic housing, and under the action of extra electric field, which will
It is mobile to cathode to carry entire solution, this phenomenon is known as electroosmotic flow.The Accurate Determining of electroosmotic flow imitates evaluation analysis method
Fruit has vital meaning, while being also the precondition for calculating some kinetic parameters.
Salomon K. establishes a kind of electroosmotic flow approximate model, and electroosmotic flow is glutinous by electric field strength, buffer in the model
Degree, buffer solution dielectric constant, separation temperature etc. influence, due to influence factor complexity, it is difficult to theoretically calculate, can only pass through
Measuring.The common methods of measurement electroosmotic flow at present are as follows: assuming that other chromatographic stationary phases of a component and capillary tube inner wall
Effect can ignore, then it is the sum of its electroosmotic flow and electrophoretic velocity that it, which closes speed,.Neutral component electrophoretic velocity is zero, that
Migration velocity is equal to the speed of electroosmotic flow.Therefore, it can choose when measuring electroosmotic flow under buffer condition used with neutrality
Component measure its transit time as marker, and then calculate to obtain electroosmotic flow.
However, such mode is very difficult for the measurement of faint electroosmotic flow, elapsed time is long, and accuracy of measurement is low.
Summary of the invention
In order to overcome the above technical problems, it the present invention provides a kind of method for measuring Capillary Electrophoresis electroosmotic flow, improves
Measurement accuracy.
To achieve the goals above, the present invention provides a kind of methods for measuring Capillary Electrophoresis electroosmotic flow, comprising:
Using the buffer dissolved with marker as sample to capillary sample introduction for the first time；
Apply first voltage and separating pressure in the capillary two sides, detects the first transit time of the marker；
By the sample to capillary sample introduction again；
Apply second voltage in the capillary two sides, while applying the separating pressure, detects the of the marker
Two transit times；Wherein the first voltage is identical as the second voltage amplitude, and polarity is opposite；
According to first transit time and second transit time, the mobility of electroosmotic flow is obtained.
Further, the sample introduction for the first time and the sample introduction again using hydrodynamic injection, vacuum sample introduction, electric power sample introduction or
Siphon sample introduction.
Further, the sample introduction for the first time 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 numberical range of the first voltage and the second voltage is 1-25000V.
Further, the separating pressure setting range is 1-99mbar.
The method of measurement Capillary Electrophoresis electroosmotic flow of the present invention, using the buffer dissolved with marker as sample
Apply first voltage and separating pressure to capillary sample introduction for the first time, and in capillary two sides, when detecting the first migration of marker
Between.Later by sample to capillary sample introduction again, apply second voltage in capillary two sides, while applying and first step operation phase
Same separating pressure, detects the second transit time of marker.Wherein first voltage is identical as second voltage amplitude, polarity phase
Instead.According to the first transit time and the second transit time, the mobility of electroosmotic flow is obtained.The scheme of the embodiment of the present invention improves electricity
The measurement accuracy of seepage flow, accuracy is higher, and is suitable for quickly measuring the faint electroosmotic flow of coatings capillary pipe, compared to biography
System method effectively saves the time, improves precision.
Detailed description of the invention
Fig. 1 is the flow chart of the method for measurement Capillary Electrophoresis electroosmotic flow of the present invention；
Fig. 2 is the schematic illustration of the method for measurement 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 measurement Capillary Electrophoresis electroosmotic flow of the present invention；
Fig. 5 is that result of the method for measurement 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 measurement Capillary Electrophoresis electroosmotic flow of the present invention under the conditions of different separation voltages
To figure；
Fig. 7 is the schematic illustration of the method for measurement Capillary Electrophoresis electroosmotic flow of the present invention.
Specific embodiment
Below with reference to the accompanying drawings illustrate the embodiment of the present invention.It is retouched in an attached drawing of the invention or a kind of embodiment
The elements and features stated can be combined with elements and features shown in one or more other drawings or embodiments.It answers
When note that for purposes of clarity, being omitted known to unrelated to the invention, those of ordinary skill in the art in attached drawing and explanation
Component or processing expression and description.
The embodiment of the invention provides a kind of methods for measuring Capillary Electrophoresis electroosmotic flow, as shown in Figure 1, including following step
It is rapid:
S101, using the buffer dissolved with marker as sample to capillary sample introduction for the first time.
Before measuring electroosmotic flow with this method, first have to select suitable neutral marker.Common selection gist is slow
Rush the type and pH value of solution, it is ensured that selected marker is in electroneutral in buffer system.Using running buffer and one
The marker for determining concentration is configured to sample.Phosphate buffer solution (the Na that specific usable pH value is 72HPO4/NaH2PO4)。
Hydrodynamic injection mode 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, specifically can be 3 seconds.Vacuum sample introduction, electric power can also be used in input mode
The modes sample introduction such as sample introduction or siphon sample introduction.
S102, apply first voltage and separating pressure in capillary two sides, detect the first transit time of marker.
The range of first voltage is 1-25000V, specifically can be 10000V.
While applying first voltage, 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 object1.In addition to UV detector, also can be used glimmering
Detectors such as photodetector, mass spectrum etc..
S103, by the sample to capillary sample introduction again.
The sampling condition of S103 is identical as S101.
S104, apply second voltage and the separating pressure in capillary two sides, detect the second transit time of marker.
Second voltage is identical as first voltage amplitude, and polarity is opposite.Apply simultaneously auxiliary with separating pressure identical in S102
Help analysis.Using the second transit time t of the recording marks object 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 indicated by formula (1):
μeo=veo/E (1)
Wherein, veoIt is electroosmotic flow speed, E is electric field strength, and E can be by V/LtIt indicates, wherein LtIt is the overall length of capillary
Degree, V are the voltage value 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 applying 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 marker under pressure, veo1For migration velocity of the marker under electroosmotic flow effect.
Apply simultaneously in above-mentioned S104, in identical capillary and the medium big pressure of S102 and equal big reversed electricity
Pressure, measuring neutral marker's transit time is t2.Then have:
Le=t2(vp2+veo2) (3)
Wherein vp2Lower marker speed, v are acted on for pressureeo2For migration velocity of the marker under electroosmotic flow effect.
Because S102, S104 are applied with, polarity is opposite but the identical voltage of size, not by the current direction of capillary
Together, size is identical, thus the Joule heat generated inside capillary in two steps 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.Meanwhile it is identical slow
System is rushed, the big voltage such as on the contrary is added, so the electroosmotic flow generated is equal big reversed.So that
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 schematic illustrations of the method for measurement capillary electroosmotic flow provided in an embodiment of the present invention.Specific electricity
Swimming condition are as follows: separation voltage: 10kV/-10kV；Separating pressure: 70mbar；Detection wavelength: 214nm；Sample introduction: 50mbar continues 3
Second；Temperature: 25 DEG C；Buffer: the phosphate buffer solution (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 the opposite still identical voltage of size of polarity, lead to
The current direction for crossing capillary is different, and size is identical, thus the Joule heat generated inside capillary in two steps 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 on the contrary, so the electroosmotic flow generated is equal big reversed.In two steps
The actual migration speed of sample is the vector sum of speed and electroosmotic flow speed that pressure is given, and pressure gives speed phase between two steps
Together, electroosmotic flow speed etc. is big reversed, then the difference of two leg speed degree is twice of electroosmotic flow speed.
Fig. 7 also illustrates the schematic illustration of the method for measurement capillary electroosmotic flow provided in an embodiment of the present invention.Two
It in secondary sample introduction, the big separating pressure in the same direction such as is applied with, and apply the opposite voltage of size identical polar, produces equal reversed 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
Measurement Capillary Electrophoresis electroosmotic flow method transit time figure.Wherein, separation voltage: 10kV/-10kV；Separating pressure:
70mbar；Detection wavelength: 214nm；Pressure when sample introduction: 50mbar, it is for 3 seconds；Temperature: 25 DEG C；Buffer: the phosphoric acid of pH7
Salt buffer solution.
Conventional method and method provided in an embodiment of the present invention all use identical sample concentration, operation architecture and sample introduction item
Part.Used neutral marker is DMSO, and concentration is 1 percent (volume ratios), and the phosphate that operation architecture is pH=7 is slow
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, being analyzed, is used using the voltage of 10kV
UV detector records sample migration time t.It carries out 5 groups to repeat to test, it is public that 5 groups of transit time is brought into following calculating respectively
Formula (8), (9),
veo=Le/t (8)
μeo=veo/ E=(LeLt)/(tV) (9)
5 groups of calculated results are averaged, the mobility as electroosmotic flow.
Method provided in an embodiment of the present invention specific steps are as follows:
Step 1: using hydrodynamic injection mode with 50mbar hydrodynamic injection 3s；
Step 2: carrying out electrophoretic analysis using 10kV voltage, while applying 70mbar pressure assistant analysis, using ultraviolet inspection
It surveys device and records sample migration time t1；
Step 3: using the sampling condition sample introduction in the first step；
Step 4: the voltage using -10kV equal with second step polarity opposite magnitude carries out electrophoretic analysis, apply simultaneously
70mbar pressure assistant analysis records sample migration time t using UV detector2。
It repeats the above steps 5 times.Multiplicating can reduce random error.
The t that 5 times are measured1、t2It brings above-mentioned formula (7) into, obtains 5 calculated results, 5 calculated results 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
From table 1, Fig. 3 and Fig. 4 it is found that comparing by new out-of-date methods, determine that the reliability of new method, new method have higher
Accuracy and repeatability.
Embodiment 2:
During electrophoretic analysis, keep other conditions it is constant, only change separating pressure, respectively 50mbar, 60mbar,
It is analyzed under the conditions of the separating pressure of 70mbar, 80mbar, 90mbar, five groups is done under the conditions of each separating pressure in parallel in fact
It tests.Probe into whether different separating pressures has an impact the efficiency of measurement electroosmotic flow.Embodiment 2 and 1 uses identical experiment
Condition, including identical long capillary tube, capillary inner diameter, operation architecture and sample.
Handled according to step documented by embodiment 1, respectively 50mbar, 60mbar, 70mbar, 80mbar,
It is analyzed under the separating pressure of 90mbar.Obtained result is as shown in table 2, Fig. 5.
Table 2
The change of the available conclusion from table 2, 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, certain correlation is not present 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 all may be used under 5 kinds of pressure
With the relatively accurate electroendosmotic mobility calculated under the deposition condition.
It, can not when selecting separating pressure although embodiment 2 illustrates that separating pressure has no influence to measurement result
Any setting, when first having to guarantee to add negative voltage, pressure is greater than the speed of electroosmotic flow to the speed of sample, and sample is enable to arrive
Up to detection window.When electrophoretic analysis, the separating pressure added is bigger, and the time needed for measuring electroosmotic flow is faster.
Embodiment 3:
It during electrophoretic analysis, keeps other conditions constant, only changes voltage, respectively in 8kV/-8kV, 10kV/-
It is 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 whether different analysis voltage has an impact the efficiency of measurement electroosmotic flow.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
The available conclusion from table 3, method measurement 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, certain correlation is not present therebetween.From fig. 6, it can be seen that in 5 kinds of voltages
Under, can be relatively accurate calculate the deposition condition under electroendosmotic mobility, 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 of Accurate Determining under this condition
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 measurement 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 of the embodiment 1 into embodiment 3, which does not pass through coating or the modification of other inner walls, capillary wall, Si-OH
Base meets buffer solution ionization, and Si-OH dissociates a H ion, keeps tube wall negatively charged, close to the buffer solution surface of tube wall
Positively charged, after applying voltage and generating electric field, solution can integrally be moved to cathode, and here it is the mechanism that electroosmotic flow generates.
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, in this way
There will be no Si-OH dissociation to lose H+Negatively charged, the solution close to capillary will not be positively charged, will not generate electroosmotic flow.
But this is perfect condition, and there are faint electroosmotic flow in coated pipe under actual conditions.If measuring the faint electricity using conventional method
Seepage flow needs 1 more than hour are 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 Biotechnology Co., Ltd.Coating
Capillary overall length 60cm, effective length 50cm, internal diameter is 75 μm, and used neutral marker is DMSO, concentration percent
One (volume ratio), operation architecture are the phosphate buffer of pH=7.
Step 1: using hydrodynamic injection mode with 50mbar hydrodynamic injection 3s；
Step 2: carrying out electrophoretic analysis using 10kV voltage, while applying 70mbar pressure assistant analysis, using ultraviolet inspection
It surveys device and records sample migration time t1；
Step 3: using sampling condition sample introduction in the first step；
Step 4: the voltage using -10kV equal with second step polarity opposite magnitude carries out electrophoretic analysis, apply simultaneously
70mbar pressure assistant analysis records sample migration time t using UV detector2。
It repeats the above steps 5 times.
The t that 5 times are measured1、t2It brings above-mentioned formula (7) into, obtains 5 calculated results, 5 calculated results 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 to quickly measurement and apply
Faint electroosmotic flow in layer pipe, and the accuracy of this method and repeatability are also proven in embodiment 1.The present invention is real
The method for measuring Capillary Electrophoresis electroosmotic flow described in example is applied to be particularly suitable for carrying out fastly the faint electroosmotic flow of coatings capillary pipe
Speed measurement, effectively saves the time compared to conventional method, improves measurement accuracy.
Although the present invention and its advantage has been described in detail it should be appreciated that without departing from by the attached claims
Defined by can carry out various changes, substitution and transformation in the case where the spirit and scope of the present invention.Moreover, the model of the application
Enclose the specific embodiment for being not limited only to process, equipment described in specification, means, method and steps.In the art is common
Technical staff is from the disclosure it will be readily understood that execution and corresponding reality described herein can be used according to the present invention
Apply the essentially identical function of example or process that obtain the result essentially identical with it, that existing and future is to be developed, equipment,
Means, method or step.Therefore, the attached claims are intended in the range of them include such process, equipment, hand
Section, method or step.
Claims (6)
1. a kind of method for measuring Capillary Electrophoresis electroosmotic flow characterized by comprising
Using the buffer dissolved with marker as sample to capillary sample introduction for the first time；
Apply first voltage and separating pressure in the capillary two sides, detects the first transit time of the marker；
By the sample to capillary sample introduction again；The sample introduction of the sampling condition of the sample introduction again and the sample introduction for the first time
Condition is identical；
Apply second voltage in the capillary two sides, while applying the separating pressure, detect the marker second moves
Shift time；Wherein the first voltage is identical as the second voltage amplitude, and polarity is opposite；
According to first transit time and second transit time, the mobility of electroosmotic flow is obtained；The mobility of the electroosmotic flow
μeo=| t1-t2|LeLt/(2t1t2V), wherein t1For the first transit time, t2For the second transit time, LeIt is effectively long for capillary
Degree, LtFor capillary total length, V is voltage value.
2. it is according to claim 1 measurement Capillary Electrophoresis electroosmotic flow method, which is characterized in that the sample introduction for the first time and
The sample introduction again uses hydrodynamic injection, vacuum sample introduction, electric power sample introduction or siphon sample introduction.
3. it is according to claim 1 measurement Capillary Electrophoresis electroosmotic flow method, which is characterized in that the sample introduction for the first time and
The sample injection time of the sample introduction again is identical with input mode.
4. the method for measurement Capillary Electrophoresis electroosmotic flow according to claim 3, which is characterized in that the sample injection time is set
It is scheduled in 1-600 seconds ranges.
5. it is according to claim 1 measurement Capillary Electrophoresis electroosmotic flow method, which is characterized in that the first voltage and
The numberical range of the second voltage is 1-25000V.
6. the method for measurement Capillary Electrophoresis electroosmotic flow according to claim 1, which is characterized in that the separating pressure
Numberical range is 1-99mbar.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US5624539A (en) * | 1995-06-19 | 1997-04-29 | The Penn State Research Foundation | Real time monitoring of electroosmotic flow in capillary electrophoresis |
| CN101021506A (en) * | 2007-03-22 | 2007-08-22 | 南京大学 | Method for measuring low-speed electric interstifial flow in chip capillary cataphoresis |
-
2017
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US5624539A (en) * | 1995-06-19 | 1997-04-29 | The Penn State Research Foundation | Real time monitoring of electroosmotic flow in capillary electrophoresis |
| CN101021506A (en) * | 2007-03-22 | 2007-08-22 | 南京大学 | Method for measuring low-speed electric interstifial flow in chip capillary cataphoresis |
Non-Patent Citations (1)
| Title |
|---|
| Method for the Accelerated Measurement of Electroosmosis in Chemically Modified Tubes for Capillary Electrophoresis;Junior E. Sandoval et al.;《Anal. Chem.》;19960715;第68卷;2771-2775 * |
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