CN101319979A - Method for fast measuring dispersion coefficient of matter in liquid phase by capillary electrophoresis apparatus - Google Patents

Abstract

The invention relates to a method for using a capillary electrophoresis instrument to detect the diffusion coefficients of a substance in a liquid phase. The invention discloses a plurality of key factors which affect the accuracy of the detection when using the capillary electrophoresis instrument to detect the diffusion coefficients of the substance, then further provides a fast detecting method which is used for the diffusion coefficients of the substance with a certain commonality and can detect the diffusion coefficients of the substance to be detected in water or an organic solvent. The method of the invention associates the diffusion coefficients of the substance with the appearance time of the elution peak of the substance as well as the width of a self-peak and can fast calculate and obtain the diffusion coefficient value of the substance.

Description

Utilize capillary electrophoresis apparatus speed to survey the method for material coefficient of diffusion in liquid phase

Technical field

The present invention relates to measure the method for material coefficient of diffusion in liquid phase with capillary electrophoresis apparatus.

Background technology

Diffusion is meant molecule, ion or the spontaneous mixing of other small-particle and by the zone of the relative high concentration process to the regional movement of low concentration.Wherein well accepted is to describe diffusion with coefficient of diffusion, and it is used for describing the change rate of concentration of the point-to-point transmission between certain distance.

Diffusion just in time show its importance at a slow speed.In most cases, diffusion occurs in sequence together with other phenomenon, and when it is in the process during the slowest step, it will become the determinative of whole process.For example, diffusion usually can limit the efficient of distillation and the reaction velocity of industrial porous catalyst, also can control the speed that the microbial growth speed that produces penicillin, speed that iron and steel is corroded and fragrance are released from food.

For biomolecule, coefficient of diffusion is an important physico-chemical property, in bioprocess very widely, and process design and analyze and all be unable to do without diffusion data.For example the protein biological respinse is analyzed, extraction and oven dry, coefficient of diffusion all is one and important parameters.In addition, the transmission performance of predicting of substance in porous medium also need be by coefficient of diffusion.In addition, coefficient of diffusion can provide people information about protein molecule size and dimension aspect in conjunction with parameters such as hydraulic performances.

Liquid Diffusion Coefficient is the research mass transport process, calculates the important foundation data of mass transfer rate and Chemical Engineering Design and exploitation, has been widely used in the emerging industries such as biochemistry and environmental protection.The coefficient of diffusion of studying and accumulate all kinds of systems is considered to be the important use fundamental research always.Yet because liquid structure is very complicated, packing of molecules density is bigger, intermolecular mean distance is little more than gas molecule, again not as good as the such regular arrangement of solid, molecule always is in the scope than strong interaction, so the measurement of Liquid Diffusion Coefficient and theoretical description are more than gas and solid difficulty, test figure quite lacks.

Measure material method of coefficient of diffusion in liquid phase and comprise methods such as membrane cisterna method, capillary tube technique, refractive index equipment method, light scattering method, nuclear magnetic resonance method, Taylor's diffusion method, fluorescence microscopy and electrochemical process.The characteristics of comprehensive these methods, comparatively speaking, the experiment of Taylor's diffusion method is simple, and equipment is simple, and measurement result is accurate, so adopted by many researchers in recent years.But Taylor's diffusion method commonly used has shortcomings such as the long and data processing of minute is loaded down with trivial details usually.Russia scholar Bello etc. has measured the coefficient of diffusion of material in liquid phase with the commodity capillary electrophoresis apparatus first, and minute was shortened in 2 minutes.But this method data processing is complicated, and lacks versatility.

Summary of the invention

The purpose of this invention is to provide a kind of commodity capillary electrophoresis apparatus of utilizing and set up a kind of new method with material coefficient of diffusion fast measuring in liquid phase of certain versatility.

When carrier with slower speed (laminar flow) when flowing through kapillary, its linear flow rate on the pipe cross section is also inequality, in the linear flow rate maximum of center capillaceous carrier, and is 0 at its linear flow rate of tube wall place.After test substance enters kapillary, the test substance molecule will flow to direction along axis of bore under the effect of carrier, its flowing velocity is also relevant with its position on the kapillary cross section, and the diffusion of molecule also makes it exert an influence in the radial and axial dispersion of kapillary.Under certain condition, the mass flux along the kapillary axial direction is the summation of diffusion part in convection current and the apparent diffusion coefficient:

$Q={\mathrm{UC}}_m+\mathrm{\δ}\frac{\∂C_m}{\∂x}---\left(1\right)$

Q is along the mass flux of tubular axis to direction in the formula, and U is the mean flow rate of carrier, C _mBe the mean concentration of test substance on tube section, δ is the apparent diffusion coefficient of test substance, is also referred to as dispersion coefficient, and x is the distance of test substance in the axial direction diffusion.

Dispersion coefficient is relevant with the coefficient of diffusion of size capillaceous, carrier flow velocity and material, and based on this, Britain scholar Aris has proposed following classical formulas:

$\mathrm{\&delta;}=D+\frac{R_c^2{\mathrm{<figure-callout\; id="2"\; label="U"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">U</figure-callout>}}^2}{48D}---\left(2\right)$

δ is the apparent diffusion coefficient of test substance in the formula, and D is the coefficient of diffusion of test substance, R _cBe inside radius capillaceous, U is the mean flow rate of carrier.If do not have mass transfer to take place at the tube wall place in the test process and be zero these two words that boundary condition all satisfies in the radially concentration gradient of tube hub place test substance, when the test substance of a pulse mode sample introduction was washed out from kapillary, then the method for available chromatogram came species distribution to be measured is carried out following expression:

$C(t,L)=A{\left[\frac{\mathrm{\&delta;}}{L^2}\right]}^{-1/2}\exp \left[\frac{-{(1-\mathrm{Ut}/L)}^2{\mathrm{<figure-callout\; id="2"\; label="L"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">L</figure-callout>}}^2}{4\mathrm{\&delta;t}}\right]---\left(3\right)$

(t is an appearance time to C in the formula for t, the L) CONCENTRATION DISTRIBUTION of expression test substance in kapillary, and L is a length capillaceous, and U is the mean flow rate of carrier, and A is the constant relevant with the test substance sample size, and δ is the apparent diffusion coefficient of test substance.Following formula is the Gaussian representation formula of a variation, if the data δ/UL in the formula (3) is enough little, following formula can be simplified to the Gaussian function of a standard.If convert the CONCENTRATION DISTRIBUTION in the formula (3) to long measure, then can be represented by the formula:

${\mathrm{\&sigma;}}^2=\frac{2\mathrm{DL}}{U}+\frac{{\mathrm{<figure-callout\; id="2"\; label="R\; c"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">R</figure-callout>}}_c^{}\mathrm{UL}}{24D}---\left(4\right)$

σ is the mean square deviation that test substance washes out the peak in the formula, and D is the coefficient of diffusion of test substance, and L is a length capillaceous, and U is the mean flow rate of carrier, R _cIt is inside radius capillaceous.And by chromatographic theory as can be known, the theoretical step height H of chromatographic peak is:

$H=\frac{{\mathrm{\&sigma;}}^2}{L}=\frac{2D}{U}+\frac{R_c^{2}U}{24D}---\left(5\right)$

σ is the mean square deviation that test substance washes out the peak in the formula, and L is a length capillaceous, and D is the coefficient of diffusion of test substance, and U is the mean flow rate of carrier, R _cIt is inside radius capillaceous.Because coefficient of diffusion is more much smaller than mean flow rate, i.e. 2D/U → 0, so following formula can be simplified to:

$H=\frac{R_c^{2}U}{24D}---\left(6\right)$

H is the theoretical step height of chromatographic peak in the formula, R _cBe inside radius capillaceous, D is the coefficient of diffusion of test substance, and U is the mean flow rate of carrier.On the other hand, theoretical step height can be tried to achieve by corresponding peak parameter, and is expressed as follows:

$H=\frac{\mathrm{<figure-callout\; id="1"\; label="L\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">L</figure-callout>}{W}_{}^{}{}_{1/2}^2}{{\mathrm{<figure-callout\; id="2"\; label="t"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}^{2}8\ln 2}---\left(7\right)$

H is the theoretical step height of chromatographic peak in the formula, and L is a length capillaceous, W _1/2Be the half-peak breadth that washes out the peak, t is the appearance time of test substance.In conjunction with above-mentioned two formulas, the computing formula of coefficient of diffusion can be simplified as follows:

$D=\frac{\mathrm{<figure-callout\; id="2"\; label="ln"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">ln</figure-callout>}2}{3}\frac{R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}=\frac{0.231R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}---\left(8\right)$

D is the coefficient of diffusion of test substance in the formula, R _cBe inside radius capillaceous, t is the appearance time of test substance, W _1/2It is the half-peak breadth that washes out the peak.Therefore, if known capillary inner diameter, then can just can obtain the coefficient of diffusion of test substance in liquid phase according to two basic peak parameter one appearance times that wash out the peak and half-peak breadth.

Method of the present invention is to be platform with a commodity capillary electrophoresis apparatus of being furnished with UV-detector, make test substance pass through kapillary with pressure, detect test substance at detection window capillaceous by UV-detector, and obtain the appearance time and the half-peak breadth at peak, thereby calculate the mensuration coefficient of diffusion of test substance with formula (8) by the workstation analysis that electrophoresis apparatus is equipped with.

Inside radius (R capillaceous _c) measured according to the Hagen-Poiseuille law, its detailed process is, in the time of 25 ℃ with certain pressure differential Δ P (60kPa＜Δ P＜150kPa) allow distilled water flow through kapillary, and collect the distilled water that washes out in 20 minutes respectively and weigh (G), then can calculate internal diameter capillaceous by formula (9).Under different pressure, measure three times and get its mean value as actual inner diameter capillaceous.

$R_c=\sqrt[4]{\frac{8\mathrm{L\&eta;Q}}{\mathrm{\&pi;}\left(\mathrm{\&Delta;P}\right)}}=\sqrt[4]{\frac{8\mathrm{L\&eta;G}}{\mathrm{\&pi;}\left(\mathrm{\&Delta;P}\right){\mathrm{gd}}_{25}t}}---\left(9\right)$

L is a total length capillaceous in the formula, and Q is the volumetric flow rate of water, and g is an acceleration of gravity, d ₂₅With η be respectively the density (0.9970mg/ml) and the viscosity (0.8909cP) of 25 ℃ of following water, t is 20 minutes.

The method of utilizing capillary electrophoresis apparatus speed to survey material coefficient of diffusion in liquid phase of the present invention, comprise to the kapillary of being furnished with UV-detector clean, the kapillary inside radius is measured, the preparation of test substance solution, detection window capillaceous by UV-detector detect that test substance carries out that Data Management Analysis, carrier flow velocity operation interval are measured, the control of test substance concentration and sampling volume controlled step:

(1) to the cleaning capillaceous of the molten silicon of being furnished with UV-detector;

(2) internal diameter capillaceous of determination step (1); With the kapillary of distilled water flushing step (1), time＞2 minute; In kapillary, have distilled water, and temperature allows distilled water flow through kapillary with 60kPa＜pressure differential Δ P＜150kPa in the time of 25 ℃, and collect the distilled water that washes out in 20 minutes respectively and weigh (G), calculate capillary radius according to formula (9) then; Get above-mentioned 3 resultant capillary radiuss of different pressures difference, and the mean value of getting them is surveyed the kapillary inside radius that the material coefficient of diffusion is used as speed;

$R_c=\sqrt[4]{\frac{8\mathrm{L\&eta;Q}}{\mathrm{\&pi;}\left(\mathrm{\&Delta;P}\right)}}=\sqrt[4]{\frac{8\mathrm{L\&eta;G}}{\mathrm{\&pi;}\left(\mathrm{\&Delta;P}\right){\mathrm{gd}}_{25}t}}---\left(9\right)$

L is a total length capillaceous in the formula, and Q is the volumetric flow rate of water, and g is an acceleration of gravity, d ₂₅With η be respectively the density 0.9970mg/ml and the viscosity 0.8909cP of 25 ℃ of following water; Δ P is a pressure differential, and t is 20 minutes;

(3) preparation of substance solution; The pattern substance solution and the certain density test substance solution that prepare a plurality of variable concentrations, and stir or method such as ultrasonic dispersing is evenly disperseed pattern material and test substance in solvent with vortex;

(4) Data Management Analysis.With the kapillary of distilled water and carrier solution rinsing step (1), the time all＞2 minutes; In kapillary, there is carrier, and the capillary temperature of step (1) is set to required temperature, choose a kind of in the pattern substance solution of a plurality of variable concentrations in the step (3), with the pattern substance solution injection kapillary of pressure with selected step (3), make the pattern substance solution pass through kapillary with pressure, at detection window capillaceous by UV-detector detecting pattern material, by capillary electrophoresis apparatus with workstation obtain appearance time and the half-peak breadth that the pattern material washes out the peak, the inside radius capillaceous that integrating step (2) is measured is directly calculated the mensuration coefficient of diffusion of pattern material by formula (8);

$D=\frac{\mathrm{<figure-callout\; id="2"\; label="ln"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">ln</figure-callout>}2}{3}\frac{R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}=\frac{0.231R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}---\left(8\right)$

D is the coefficient of diffusion of pattern material in the formula, R _cBe inside radius capillaceous, t is the appearance time of pattern material, W _1/2It is the half-peak breadth that washes out the peak;

Repeating step (4) step is until the mensuration coefficient of diffusion of the pattern material that obtains a plurality of variable concentrations;

(5) choose a kind of in the pattern substance solution of a plurality of variable concentrations in the step (3) and come determination step (1) carrier flow velocity capillaceous operation interval; With the kapillary of distilled water and carrier solution rinsing step (1), the time all＞2 minutes; In kapillary, there is carrier, the capillary temperature of step (1) is set to required temperature, with the pattern substance solution injection kapillary of pressure with selected step (3), select for use different pressures to make the pattern material pass through kapillary respectively, at detection window capillaceous by UV-detector detecting pattern material, coefficient of diffusion under the carrier flow velocity of mode determination material in step (1) kapillary, with the carrier flow velocity coefficient of diffusion is mapped then, coefficient of diffusion of measuring among the figure and selected step (3) known mode material actual dispersion coefficient are the operation interval of carrier flow velocity near the zone of (deviation is less than 2%).If change the kapillary of step (1), then must redefine the kapillary operation interval of step (1).

(6) kapillary of usefulness distilled water and carrier solution rinsing step (1), time is equal＞and 2 minutes, in kapillary, there is carrier, the capillary temperature of step (1) is set to required temperature, the pattern substance solution of a plurality of variable concentrations of step (3) is injected kapillary respectively; Flow velocity under a certain pressure in the operation interval of the carrier flow velocity that optional step (5) is determined, make above-mentioned pattern material pass through kapillary respectively, at detection window capillaceous by UV-detector detecting pattern material, coefficient of diffusion under the mode determination material variable concentrations, coefficient of diffusion with said determination is mapped to its corresponding pattern material concentration respectively, and the coefficient of diffusion of mensuration and known mode material actual dispersion coefficients deviation are the Cmax of pattern material greater than 2% pattern material concentration.

(7) kapillary of usefulness distilled water and carrier solution rinsing step (1), time is equal＞and 2 minutes, in kapillary, there is carrier, the capillary temperature of step (1) is set to required temperature, and the pattern substance solution that will be lower than any concentration of the measured max model material concentration of step (6) injects kapillary; Flow velocity under a certain pressure in the operation interval of the carrier flow velocity that optional step (5) is determined makes above-mentioned pattern material by kapillary, and the coefficient of diffusion of mode determination material under different sampling volumes mapped to coefficient of diffusion with pattern material sampling volume then.Coefficient of diffusion of measuring and known mode material actual dispersion coefficients deviation are the pattern material greater than 2% sampling volume maximum sampling volume;

(8) with the kapillary of distilled water and carrier solution rinsing step (1), the time all＞2 minutes; In kapillary, there is carrier, and the capillary temperature of step (1) is set to required temperature, choose a kind of test substance solution in the step (3), with the test substance solution injection kapillary that be lower than step (6) measured max model material concentration of pressure with selected step (3) preparation, and make its sampling volume less than the measured maximum sampling volume of step (7), flow velocity under a certain pressure in the operation interval of the carrier flow velocity that optional step (5) is determined washes out test substance, detect test substance at detection window capillaceous by UV-detector, by capillary electrophoresis apparatus with workstation get appearance time and the half-peak breadth that certain density test substance washes out the peak, the inside radius capillaceous that integrating step (2) is measured is directly calculated the mensuration coefficient of diffusion of test substance by formula (8);

$D=\frac{\mathrm{<figure-callout\; id="2"\; label="ln"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">ln</figure-callout>}2}{3}\frac{R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}=\frac{0.231R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}---\left(8\right)$

D is the coefficient of diffusion of test substance in the formula, R _cBe inside radius capillaceous, t is the appearance time of test substance, W _1/2It is the half-peak breadth that washes out the peak.

The described solvent of step (3) is the carrier of pattern material or test substance.

Described carrier is distilled water or organic solvent.As, when measuring certain test substance coefficient of diffusion in water, should with water carrier then; Coefficient of diffusion in certain organic solvent should be a carrier with this organic solvent then.

Described test substance is to be dissolved in distilled water or organic solvent, perhaps can evenly disperse to form the material of thermodynamic stable system (as colloidal gold solution) in water or organic solvent.

The described cleaning of step (1) is to use washed with methanol＞10 minute successively, and distilled water cleaned＞5 minutes, and the nitric acid of 1mol/L cleaned＞10 minutes, and distilled water cleaned＞5 minutes, and the NaOH of 1mol/L cleaned＞10 minutes, uses distilled water flushing＞5 minute at last.Between per twice measurement, kapillary washed＞5 minutes with 1mol/L NaOH.

The pattern substance solution of a plurality of variable concentrations of the described preparation of step (3) is 3 above pattern substance solutions.

Step (5) is described with the pattern substance solution injection kapillary of pressure with selected step (3), and selects for use different pressures to make the pattern material by kapillary respectively, and its pressure limit all is 0.69～137.90kPa.

It is described that to select for use different pressures to make the pattern material respectively be to choose 6 above spot pressures by pressure capillaceous.

Step (4) is described with the pattern substance solution injection kapillary of pressure with selected step (3), and makes the pattern substance solution by kapillary with pressure, and its pressure limit all is 0.69～137.90kPa.

It is 0.69～137.90kPa that the described test substance solution that is lower than the measured max model material concentration of step (6) that selected step (3) is prepared with pressure of step (8) injects pressure capillaceous.

The invention discloses a kind of method of utilizing the commodity capillary electrophoresis apparatus material coefficient of diffusion in liquid phase to be carried out rapid and accurate determination.The present invention has disclosed and has utilized capillary electrophoresis apparatus to measure test substance in liquid phase during coefficient of diffusion, the multiple key factor of accuracy is measured in influence, and then set up a kind of rapid assay methods, and can measure the coefficient of diffusion of material in water or non-aqueous solvent with material coefficient of diffusion in liquid phase of certain versatility.The present invention associates the coefficient of diffusion of material washes out the peak with it appearance time and half-peak breadth, can calculate its coefficient of diffusion value fast.With respect to the existing method of measuring the material coefficient of diffusion, it has fast, micro-, accurately reach advantages such as general.

Under the situation of the spirit and scope of the present invention, the present invention can make many various enforcements, so should be understood that, except that the right requirement limited, the present invention was not limited to the specific embodiment that this paper provides.

Description of drawings

Fig. 1. the graph of a relation between the carrier flow velocity of the embodiment of the invention 1 and the L-phenylalanine coefficient of diffusion that records.

Fig. 2. the relation between the pressure of the embodiment of the invention 1 and carrier flow velocity.

Fig. 3. the graph of a relation between the sampling volume of the L-phenylalanine of the embodiment of the invention 1 and the coefficient of diffusion that records.

Fig. 4. the graph of a relation between the sampling volume of the embodiment of the invention 1 and the L-phenylalanine coefficient of diffusion that records.

Embodiment

The radius that provides with a producer is that 37.5 μ m, length are that the kapillary of 60cm (the sample introduction end is 50cm to the distance of detection window) is the selection that example illustrates condition determination.(Mcgraw given L-phenylalanine in its works " American institute of physics handbook " book is 7.06 * 10 in the value of 25 ℃ of coefficient of diffusion with the L-phenylalanine during mensuration ^-6Cm ²/ s) be example.By following detailed description with the accompanying drawing, purpose of the present invention and method can better be understood.

(1) molten silicon cleaning capillaceous.Clean kapillary according to following steps: washed with methanol 15 minutes, water cleaned 10 minutes, and the nitric acid of 1mol/L cleaned 15 minutes, and water cleaned 10 minutes, and the NaOH of 1mol/L cleaned 15 minutes, final rinse water 10 minutes.Between per twice measurement, kapillary washed 5 minutes with 1mol/L NaOH, and water and carrier solution respectively washed 2 minutes then.

(2) internal diameter capillaceous of determination step (1).Measure internal diameter capillaceous according to the Hagen-Poiseuille law.In the time of 25 ℃, with 67.33kPa, 100.66kPa and the pressure differential of 135.43kPa allows distilled water flow through kapillary, and collect the distilled water that washes out in 20 minutes respectively and weigh (G), calculate capillary radius respectively according to formula (9) then, get their mean value and survey the kapillary inside radius that the material coefficient of diffusion is used as speed;

$R_c=\sqrt[4]{\frac{8\mathrm{L\&eta;Q}}{\mathrm{\&pi;}\left(\mathrm{\&Delta;P}\right)}}=\sqrt[4]{\frac{8\mathrm{L\&eta;G}}{\mathrm{\&pi;}\left(\mathrm{\&Delta;P}\right){\mathrm{gd}}_{25}t}}---\left(9\right)$

L is a total length capillaceous in the formula, and Q is the volumetric flow rate of water, and g is an acceleration of gravity, d ₂₅With η be respectively the density 0.9970mg/ml and the viscosity 0.8909cP of 25 ℃ of following water; Δ P is a pressure differential, and t is 20 minutes; The radius that records is respectively 36.36,36.31 and 36.34 μ m, and getting its mean value 36.34 μ m is inside radius capillaceous.

(3) preparation of substance solution; Preparation concentration is respectively 1.21,2.42,3.63,4.84,6.05,12.10,24.21,36.32,48.43 and the aqueous solution of the L-phenylalanine (pattern material) of 60.53mM and acetone, phenol, benzene, the aqueous solution of toluene and the ethanol and 1-butanols (test substance) solution of benzene that concentration is 2mM.

(4) Data Management Analysis.With the kapillary of distilled water flushing step (1), the time is 2 minutes; In kapillary, there is distilled water, and the capillary temperature of step (1) is set to 25 ℃, choose a kind of in the L-phenylalanine solution of a plurality of variable concentrations in the step (3), with the pressure of 1.34kPa the L-phenylalanine solution of selected step (3) is injected kapillary, make L-phenylalanine solution respectively with 0.02264 with the pressure that is selected from 0.69～137.90kPa, 0.04304,0.10482,0.21313,0.60386,0.94697,1.24378,1.51515,1.75439 and the distilled water flow velocity of 2.12585cm/s passes through kapillary, detect the L-phenylalanine at detection window capillaceous by UV-detector, by capillary electrophoresis apparatus with workstation obtain appearance time and the half-peak breadth that the L-phenylalanine washes out the peak, the inside radius capillaceous that integrating step (2) is measured is directly calculated the mensuration coefficient of diffusion of L-phenylalanine by formula (8);

$D=\frac{\mathrm{<figure-callout\; id="2"\; label="ln"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">ln</figure-callout>}2}{3}\frac{R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}=\frac{0.231R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}---\left(8\right)$

D is the coefficient of diffusion of L-phenylalanine in the formula, R _cBe inside radius capillaceous, t is the appearance time of L-phenylalanine, W _1/2It is the half-peak breadth that washes out the peak;

Repeating step (4) step is until the mensuration coefficient of diffusion of the L-phenylalanine that obtains a plurality of variable concentrations;

(5) choosing concentration in the step (3) is that the L-phenylalanine aqueous solution of 1.21mM is come determination step (1) carrier flow velocity capillaceous operation interval; With the kapillary of distilled water solution rinsing step (1), the time is 2 minutes; In kapillary, there is distilled water, the capillary temperature of step (1) is set to required temperature (25 ℃), with the pressure of 1.34kPa the L-phenylalanine solution of selected step (3) is injected kapillary, with the pressure that is selected from 0.69～137.90kPa measure respectively its in step (1) kapillary 0.02264,0.04304,0.10482,0.21313,0.60386,0.94697,1.24378,1.51515,1.75439 and the coefficient of diffusion under the distilled water flow velocity of 2.12585cm/s, with the distilled water flow velocity coefficient of diffusion is mapped then, coefficient of diffusion that records among the figure and L-phenylalanine actual dispersion coefficient are the operation interval of distilled water flow velocity near the zone of (deviation is less than 2%).

As shown in Figure 1 in 0.1 to 1cm/s distilled water flow rates, the deviation minimum of the coefficient of diffusion that records, that is to say that horizontal component among the figure (area I I, it comprises two parts up and down of horizontal dotted line) is effective operation interval of accurately measuring the material coefficient of diffusion under selected kapillary.Horizontal dotted line among Fig. 1 is represented the actual dispersion coefficient value of L-phenylalanine under this temperature.And in zone (it comprises two parts up and down of horizontal dotted line I) and area I II (it comprises two parts up and down of horizontal dotted line I), the coefficient of diffusion value of the L-phenylalanine that records is more on the low side and higher than its actual value respectively.Therefore, when the inside radius that uses is 36.34 μ m, length when measuring the coefficient of diffusion of material as the kapillary of 60cm (effective length 50cm), the distilled water flow velocity should be controlled at 0.1～1cm/s (operation interval).

In the present invention, distilled water is mobile by pressure-driven, and as seen from Figure 2, in the pressure limit of 40kPa, the pressure differential at the flow velocity of distilled water and kapillary two ends is linear.And flow rate corresponding is 1.00cm/s under the pressure of 40kPa, and this value also is accurately to measure the upper limit of pressure of coefficient of diffusion.After surpassing this upper pressure limit, curve begins to be bent downwardly, and the coefficient of diffusion value that records is bigger than normal.In addition, this test also provides a kind of mensuration experimental technique of high effective liquidate speed.

Working range that it should be noted that flow velocity is also relevant with length capillaceous and internal diameter.For example, when to use a radius be 12.14 μ m, effective length as the kapillary of 50cm, the working range of its flow velocity shortened to 0.1～0.2cm/s; And when a radius be that 35.85 μ m, effective length are the kapillary of 20cm when being used to measure the coefficient of diffusion of material, the working range of distilled water flow velocity also has only 0.3～0.7cm/s.Therefore, behind the kapillary that more renews, the working range of flow velocity needs to demarcate again, and internal diameter capillaceous also need redeterminate.

(6) kapillary of usefulness distilled water flushing step (1), time is 2 minutes, in kapillary, there is distilled water, the capillary temperature of step (1) is set to required temperature (25 ℃), the L-phenylalanine solution of a plurality of variable concentrations of step (3) is injected kapillary respectively; The flow velocity of 0.20cm/s in the operation interval of the distilled water flow velocity that optional step (5) is determined, make the L-phenylalanine pass through kapillary, detect the L-phenylalanine at detection window capillaceous by UV-detector, measure the coefficient of diffusion under the L-phenylalanine variable concentrations, coefficient of diffusion with said determination is mapped (as shown in Figure 3) to its corresponding L-concentration of phenylalanine respectively, the coefficient of diffusion deviation that records is 6.05mM greater than 2% L-concentration of phenylalanine, and this concentration is the Cmax of L-phenylalanine.

(7) kapillary of usefulness distilled water flushing step (1), time is 2 minutes, in kapillary, there is distilled water, the capillary temperature of step (1) is set to required temperature (25 ℃), and the L-phenylalanine aqueous solution that will be lower than the 1.21mM of the measured max model material concentration of step (6) is injected kapillary; The distilled water flow velocity of 0.20cm/s in the operation interval of the distilled water flow velocity that optional step (5) is determined, make the L-phenylalanine pass through kapillary, measure the coefficient of diffusion of L-phenylalanine under different sampling volumes, with L-phenylalanine sampling volume coefficient of diffusion is mapped (as shown in Figure 4) then.The deviation of the coefficient of diffusion that records is 10nL greater than 2% sampling volume, and this is the maximum sampling volume of test substance;

(8) with the kapillary of distilled water and carrier solution rinsing step (1), the time is 2 minutes; In kapillary, there is carrier (corresponding to the solvent in the table 1), and the capillary temperature of step (1) is set to required temperature (corresponding to the temperature in the table 1), choose a kind of test substance in the step (3) (corresponding to the solute in the table 1) solution, pressure with 1.34kPa injects kapillary with the test substance solution that is lower than the measured L-phenylalanine Cmax of step (6) that selected step (3) prepares, and to make its sampling volume be 4.5nL, less than the measured maximum sampling volume of step (7), the flow velocity of 0.20cm/s in the operation interval of carrier (corresponding to the carrier in the table 1) flow velocity that optional step (5) is determined washes out test substance (corresponding to the solute in the table 1), detect test substance (corresponding to the solute in the table 1) at detection window capillaceous by UV-detector, by capillary electrophoresis apparatus with workstation get appearance time and the half-peak breadth that test substance (corresponding to the solute in the table 1) washes out the peak, the inside radius capillaceous that integrating step (2) is measured is directly calculated the mensuration coefficient of diffusion (corresponding to the measured value in the table 1) of test substance by formula (8);

$D=\frac{\mathrm{<figure-callout\; id="2"\; label="ln"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">ln</figure-callout>}2}{3}\frac{R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}=\frac{0.231R_c^2\mathrm{<figure-callout\; id="1"\; label="t\; W"\; filenames="A20071010007500162.png"\; state="\{\{state\}\}">t</figure-callout>}}{W_{}^{}}---\left(8\right)$

D is the coefficient of diffusion of test substance (corresponding to the solute in the table 1) in the formula, R _cBe inside radius capillaceous, t is the appearance time of test substance (corresponding to the solute in the table 1), W _1/2It is the half-peak breadth that washes out the peak.

Table 1 is the coefficient of diffusion value (corresponding to the measured value of table 1) of a series of test substances (corresponding to the solute in the table 1) in water or organic solvent that records, compare with literature value (corresponding to the literature value of table 1), measurement result accuracy height, measurement deviation is all in 2%.

Table 1

Claims (10)

1. one kind is utilized the fast method of surveying material coefficient of diffusion in liquid phase of capillary electrophoresis apparatus, comprise to the kapillary of being furnished with UV-detector clean, the kapillary inside radius is measured, the preparation of test substance solution, detection window capillaceous by UV-detector detect that test substance carries out that Data Management Analysis, carrier flow velocity operation interval are measured, the control of test substance concentration and sampling volume controlled step, it is characterized in that:

(1) to the cleaning capillaceous of the molten silicon of being furnished with UV-detector;

(2) with the kapillary of distilled water flushing step (1), and temperature allows distilled water flow through kapillary with 60kPa＜Δ P＜150kPa in the time of 25 ℃, and collects the distilled water that washes out in 20 minutes respectively and weigh, and calculates capillary radius according to formula (9) then; Get above-mentioned 3 resultant capillary radiuss of different pressures difference, and the mean value of getting them is surveyed the kapillary inside radius that the material coefficient of diffusion is used as speed;

$R_c=\sqrt[4]{\frac{8\mathrm{L\&eta;Q}}{\mathrm{\&pi;}\left(\mathrm{\&Delta;P}\right)}}=\sqrt[4]{\frac{8\mathrm{L\&eta;G}}{\mathrm{\&pi;}\left(\mathrm{\&Delta;P}\right)g{d}_{25}t}}---\left(9\right)$

L is a total length capillaceous in the formula, and Q is the volumetric flow rate of water, and g is an acceleration of gravity, and G is the distilled water weight of collecting in 20 minutes, d ₂₅With η be respectively the density 0.9970mg/ml and the viscosity 0.8909cP of 25 ℃ of following water; Δ P is a pressure differential, and t is 20 minutes;

(3) the pattern substance solution and the certain density test substance solution of a plurality of variable concentrations of preparation;

(4) kapillary of usefulness distilled water and carrier solution rinsing step (1), in kapillary, there is carrier, and the capillary temperature of step (1) is set to required temperature, choose a kind of in the pattern substance solution of a plurality of variable concentrations in the step (3), inject kapillary and pass through kapillary with the pattern substance solution of pressure selected step (3), at detection window capillaceous by UV-detector detecting pattern material, by capillary electrophoresis apparatus with workstation obtain appearance time and the half-peak breadth that the pattern material washes out the peak, the inside radius capillaceous that integrating step (2) is measured is directly calculated the mensuration coefficient of diffusion of pattern material by formula (8);

$D=\frac{\ln 2}{3}\frac{R_c^{2}t}{W_{1/2}^2}=\frac{0.231R_c^{2}t}{W_{1/2}^2}---\left(8\right)$

D is the coefficient of diffusion of pattern material in the formula, R _cBe inside radius capillaceous, t is the appearance time of pattern material, W _1/2It is the half-peak breadth that washes out the peak;

Repeating step (4) step is until the mensuration coefficient of diffusion of the pattern material that obtains a plurality of variable concentrations;

(5) choose a kind of in the pattern substance solution of a plurality of variable concentrations in the step (3) and come determination step (1) carrier flow velocity capillaceous operation interval; Kapillary with distilled water and carrier solution rinsing step (1), in kapillary, there is carrier, the capillary temperature of step (1) is set to required temperature, with the pattern substance solution injection kapillary of pressure with selected step (3), select for use different pressures to make the pattern material pass through kapillary respectively, at detection window capillaceous by UV-detector detecting pattern material, coefficient of diffusion under the carrier flow velocity of mode determination material in step (1) kapillary, with the carrier flow velocity coefficient of diffusion is mapped then, coefficient of diffusion of measuring among the figure and selected step (3) known mode material actual dispersion coefficients deviation are the operation interval of carrier flow velocity less than 2% zone;

(6) with the kapillary of distilled water and carrier solution rinsing step (1), in kapillary, there is carrier, the capillary temperature of step (1) is set to required temperature, the pattern substance solution of a plurality of variable concentrations of step (3) is injected kapillary respectively; Flow velocity under a certain pressure in the operation interval of the carrier flow velocity that optional step (5) is determined, make above-mentioned pattern material pass through kapillary respectively, at detection window capillaceous by UV-detector detecting pattern material, coefficient of diffusion under the mode determination material variable concentrations, coefficient of diffusion with said determination is mapped to its corresponding pattern material concentration respectively, and the coefficient of diffusion of mensuration and known mode material actual dispersion coefficients deviation are the Cmax of pattern material greater than 2% pattern material concentration;

(7) kapillary of usefulness distilled water and carrier solution rinsing step (1), in kapillary, there is carrier, the capillary temperature of step (1) is set to required temperature, and the pattern substance solution that will be lower than any concentration of the measured max model material concentration of step (6) injects kapillary; Flow velocity under a certain pressure in the operation interval of the carrier flow velocity that optional step (5) is determined makes above-mentioned pattern material by kapillary, and the coefficient of diffusion of mode determination material under different sampling volumes mapped to coefficient of diffusion with pattern material sampling volume then; Coefficient of diffusion of measuring and known mode material actual dispersion coefficients deviation are the pattern material greater than 2% sampling volume maximum sampling volume;

(8) kapillary of usefulness distilled water and carrier solution rinsing step (1), in kapillary, there is carrier, and the capillary temperature of step (1) is set to required temperature, choose a kind of test substance solution in the step (3), with the test substance solution injection kapillary that be lower than step (6) measured max model material concentration of pressure with selected step (3) preparation, and make its sampling volume less than the measured maximum sampling volume of step (7), flow velocity under a certain pressure in the operation interval of the carrier flow velocity that optional step (5) is determined washes out test substance, detect test substance at detection window capillaceous by UV-detector, by capillary electrophoresis apparatus with workstation get appearance time and the half-peak breadth that certain density test substance washes out the peak, the inside radius capillaceous that integrating step (2) is measured is directly calculated the mensuration coefficient of diffusion of test substance by formula (8);

$D=\frac{\ln 2}{3}\frac{R_c^{2}t}{W_{1/2}^2}=\frac{0.231R_c^{2}t}{W_{1/2}^2}---\left(8\right)$

D is the coefficient of diffusion of test substance in the formula, R _cBe inside radius capillaceous, t is the appearance time of test substance, W _1/2It is the half-peak breadth that washes out the peak.

2. method according to claim 1 is characterized in that: described test substance is to be dissolved in distilled water or organic solvent, perhaps can evenly disperse to form the material of thermodynamic stable system in water or organic solvent.

3. method according to claim 1 is characterized in that: the described solvent of step (3) is the carrier of pattern material or test substance.

4. according to claim 1 or 3 described methods, it is characterized in that: described carrier is distilled water or organic solvent.

5. method according to claim 1 is characterized in that: the described cleaning of step (1) is to use washed with methanol successively, and distilled water cleans, and nitric acid cleans, and distilled water cleans, and NaOH cleans, and uses distilled water flushing at last.

6. method according to claim 1 is characterized in that: the pattern substance solution of a plurality of variable concentrations of the described preparation of step (3) is 3 above pattern substance solutions.

7. method according to claim 1, it is characterized in that: step (5) is described with the pattern substance solution injection kapillary of pressure with selected step (3), and select for use different pressures to make the pattern material respectively by kapillary, its pressure limit all is 0.69～137.90kPa.

8. method according to claim 7 is characterized in that: described to select for use different pressures to make the pattern material respectively be to choose 6 above spot pressures by pressure capillaceous.

9. method according to claim 1, it is characterized in that: step (4) is described with the pattern substance solution injection kapillary of pressure with selected step (3), and making the pattern substance solution by kapillary with pressure, its pressure limit all is 0.69～137.90kPa.

10. method according to claim 1 is characterized in that: it is 0.69～137.90kPa that the described test substance solution that is lower than the measured max model material concentration of step (6) that selected step (3) is prepared with pressure of step (8) injects pressure capillaceous.

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