CN108279222A - A kind of method that dual wavelength resonance rayleigh light scattering method measures chitosan content - Google Patents
A kind of method that dual wavelength resonance rayleigh light scattering method measures chitosan content Download PDFInfo
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Abstract
The present invention relates to a kind of methods that dual wavelength resonance rayleigh light scattering method measures chitosan content, belong to big Molecular Detection field.In the prior art the content of chitosan in finished product is difficult to quantify to overcome, the cumbersome technical deficiency of operating technology, the present invention provides a kind of method that dual wavelength resonance rayleigh light scattering method measures chitosan content, the present invention utilizes under certain condition, brilliant blue dyestuff is combined with chitosan, a kind of ionic associate that the Resonance Rayleigh Scattering signal that can make in system is remarkably reinforced is formed, and occurs two characteristic peaks at 345nm and 445nm.Under conditions selected, chitosan concentration has good linear relationship within the scope of 0.005 1.8 μ g/mL with resonance Rayleigh intensity, establishes a kind of dual wavelength resonance rayleigh light scattering method measuring chitosan content accordingly.This method measurement influenced by the molecular weight of chitosan, have reagent it is cheap, high sensitivity, favorable reproducibility, strong antijamming capability and it is easy to operate the advantages that.
Description
Technical field
The present invention relates to a kind of methods that dual wavelength resonance rayleigh light scattering method measures chitosan content, belong to big Molecular Detection
Field.
Background technology
Chitin (chitin) also known as chitin are by N- acetyl -2-amino-2-deoxy-D-Glucose with β-Isosorbide-5-Nitrae glucosides
The polysaccharide that key-shaped formula is formed by connecting.It is widely present in the shell of crustacean shrimp, crab and insect etc., the low plant in nature
Object mushroom, algae cell, the cell wall etc. of higher plant.Up to 100,000,000,000 tons of the amount of the annual biosynthesis of chitin, is deposited
Amount is only second to cellulose in nature.
Chitosan (chitosan, CTS) is the substance that linear glycosaminoglycan chitin is generated by deacetylation, point
Many amino, hydroxyl and deacetylation are dispersed in subchain, molecular weight is differed from hundreds of thousands to millions of, in aqueous solution shape
At various intramoleculars and intermolecular hydrogen bonding, and these hydrogen bonds make chitosan form hydrophobic microcell, and then form its double-spiral structure.
Chitosan in an acidic solution, forms positively charged ion after amino absorption proton, can pass through with negatively charged anion
Electrostatical binding and hydrophobic effect form associated matter.The good characteristic of chitosan is used in medicine and pharmacology, health food, Shui Chu respectively
Having in all conglomeraties such as reason, METAL EXTRACTION and recycling and has been widely used, the especially application in terms of health food is gradually extensive,
Therefore the content of Accurate Determining chitosan is of great significance for research and development chitosan.Currently, the survey of chitosan content
The method of determining has four class such as spectroscopic methodology, electrochemical process, titration, chromatography.
Resonance Rayleigh Scattering (Resonance Rayleigh Scattering, RRS) method refer to when Rayleigh scattering is located at or
When person is close to its molecular absorption band, Electron absorption wave frequency is identical as scattering frequency, and electronics is strong because of resonance
Light absorbing energy simultaneously generates scattering again, and this absorb-again scattering process be known as Resonance Rayleigh Scattering, and it is due to scattering
Intensity improves several orders of magnitude than simple Rayleigh scattering, therefore no longer follows Rayleigh's law.Resonance rayleigh light scattering method is in recent years
Come the high sensitivity just to grow up, Molecular spectral analytical methods easy to operate.Resonance rayleigh light scattering method is answered extensively at present
For the analysis of large biological molecule, inorganic matter and Pharmaceutical Analysis, the measurement of trace inorganic ion and nanoparticle characterization etc.
Research.Its method is applied to the analysis of glucide herein, establishes the Resonance Rayleigh Scattering new method for measuring chitosan, and grind
Study carefully it and reacts suitable condition and its influence factor.
Brilliant blue (Brilliant blue) is a kind of anionic dye, under mild acid conditions, negatively charged brilliant blue and band
Ion association reaction occurs for the chitosan of positive charge, and ionic associate generates two new characteristic peaks at 345nm, 445nm,
And within the scope of a certain concentration, scattering strength is in a linear relationship with chitosan content.According to this characteristic, a kind of anti-interference energy is established
The strong resonance rayleigh light scattering method of power, the method are suitble to measure the quantitative analysis for chitosan content in health products.
Invention content
In the prior art the content of chitosan in finished product is difficult to quantify to overcome, the cumbersome technology of operating technology is not
Foot, the present invention provide a kind of method that dual wavelength resonance rayleigh light scattering method measures chitosan content, and the present invention utilizes certain condition
Under, brilliant blue dyestuff is combined with chitosan, and a kind of ion that the Resonance Rayleigh Scattering signal that can make in system is remarkably reinforced of formation is formed
Object is closed, and occurs two characteristic peaks at 345nm and 445nm.Under conditions selected, chitosan concentration is in 0.005-1.8 μ g/
There is good linear relationship with resonance Rayleigh intensity within the scope of mL, establishes a kind of double wave measuring chitosan content accordingly
Long resonance rayleigh light scattering method.This method measurement is influenced by the molecular weight and deacetylation of chitosan, has reagent cheap, sensitive
Degree is high, favorable reproducibility, strong antijamming capability and it is easy to operate the advantages that.
A kind of method that dual wavelength resonance rayleigh light scattering method measures chitosan content comprising following step：
1) standard curve of the chitosan concentration of Δ I and different molecular weight is drawn
The chitosan standard solution of addition 1.0mL a certain concentration gradients into 10mL colorimetric cylinders, 1.0mL buffer solutions,
1.0mL a concentration of 1.0 × 10-3The brilliant blue solution of mol/L is settled to scale using distilled water three times, and fully shakes up, and is set
In room temperature room temperature 10min, take 1ml mixed solutions that quartz colorimetric utensil is added, on F-2500 type sepectrophotofluorometers, with λex=
λemScanning is synchronized, respectively each detection architecture of record chitosan-containing body at maximum resonance Rayleigh scattering wavelength X=345nm
The resonance scattering intensity value I of system1, the wherein resonance scattering intensity value I of reagent blank system at λ=345nm01, and calculate Δ
I1=I1-I01；Simultaneously record chitosan-containing each detection architecture resonate at secondary big Resonance Rayleigh Scattering wavelength X=445nm it is auspicious
Sharp scattering strength I2With the Resonance Rayleigh Scattering intensity I of blank reagent02, calculate Resonance Rayleigh Scattering strength difference Δ I2=I2-
I02, wherein Δ I=Δs I1+ΔI2.Establish the standard curve C1 of Δ I and low molecular chitosan concentration；It is poly- using middle-molecular-weihydroxyethyl shell
Sugar juice replaces low molecular chitosan solution, establishes the standard curve C2 of Δ I and middle molecular chitosan concentration；Use high molecular weight
Chitosan solution replaces low molecular chitosan solution, establishes the standard curve C3 of Δ I and polymer chitosan concentration；
Under the best experimental conditions, measurement result of the system to the chitosan of different molecular weight is investigated.It investigates respectively
The chitosan of low molecular weight, middle-molecular-weihydroxyethyl and high molecular weight.By statistical analysis, the chitosan result of different molecular weight is deposited
In the significance difference opposite sex, this method measures chitosan content to be influenced by different molecular weight.
2) preparation of the sample working solution of 10 μ g/mL：Chitosan capsules are gone into capsule shells, weigh 0.04g in 100mL capacity
It in bottle, is dissolved with 0.5mol/L glacial acetic acid, constant volume obtains stock sample solution.Storing solution, filtrate warp are filtered with funnel absorbent cotton
6000r/min, centrifuge 20min, take supernatant 2.5mL in 100mL volumetric flasks, and constant volume obtains the sample of a concentration of 10 μ g/mL
Product working solution.
3) according to chitosan molecule amount selection criteria curve：Δ I is drawn using detection method in step 1) and sample works
The standard curve of liquid, and by it compared with the chitosan standard curve of different molecular weight, according to the Δ I and sample working solution of sample
Standard curve regression equation determine the chitosan in sample molecular weight size, according to the molecule of corresponding chitosan
The size of amount determines used regression equation；
4) chitosan content determines in sample：Sample working solution 1mL is taken, is measured by step 1) detection method, in wave
Its Resonance Rayleigh Scattering strength difference Δ I is measured at long 345nm and 445nm1With Δ I2, and calculate Δ I=Δs I1+ΔI2, by Δ
I substitutes into equation of linear regression and acquires the content of chitosan in sample, while doing mark-on reclaims experiment.
The method that a kind of dual wavelength resonance rayleigh light scattering method as described above measures chitosan content, the buffer solution are B-
In R buffer solutions, HAc-NaAc buffer solutions, glycine-HCI buffer solution, citrate-phosphate disodium hydrogen buffer solution
It is a kind of；Applicants experimentally found that the acidity of buffer solution has significant impact to the reaction system, in pH=3.0
Scattering value is larger；And for system in citric acid-sodium citrate buffer medium sensitivity highest, scattering value is maximum, therefore best slow
It rushes solution and is selected as pH=3.0 citrate-phosphate disodium hydrogen buffer solutions.
The method that a kind of dual wavelength resonance rayleigh light scattering method as described above measures chitosan content, reagent in step 1
Addition sequence is to be firstly added citrate-phosphate disodium hydrogen buffer solution, and brilliant blue solution is secondly added, dodecyl is added again
Sodium sulphate anionic surfactant solution, is eventually adding chitosan solution.
The method that a kind of dual wavelength resonance rayleigh light scattering method as described above measures chitosan content, chitosan in step 1)
Concentration range be 0.005-1.8 μ g/mL.
Sample pre-treatments：Chitosan capsules are gone into capsule shells, weigh 0.04g in 100mL volumetric flasks, with 0.5mol/L ice
Acetate dissolution, constant volume obtain stock sample solution.Storing solution is filtered with funnel absorbent cotton, filtrate is through 6000r/min, centrifuge
20min, takes supernatant 2.5mL in 100mL volumetric flasks, and constant volume obtains the sample working solution of a concentration of 10 μ g/mL.
Sample working solution 1mL is taken, is measured by step 1) detection method, it is total that it is measured at wavelength 345nm and 445nm
Rayleigh intensity of shaking difference DELTA I1With Δ I2, and calculate Δ I=Δs I1+ΔI2, Δ I is substituted into equation of linear regression and acquires sample
The content of chitosan in product, while doing mark-on reclaims experiment.
Test example：
1. the Resonance Rayleigh Scattering collection of illustrative plates of brilliant blue-chitosan system
After preparing solution according to experimental method, in synchronous scanning Resonance Rayleigh Scattering on F-2500 type sepectrophotofluorometers
Collection of illustrative plates (such as Fig. 1).Fig. 1 is the blank tube of brilliant blue solution-buffer solution and the different amounts of chitosan and being total to for brilliant blue associated matter
It shakes Rayleigh Scattering Spectra, the results showed that：Brilliant blue solution is weaker (bent in determination condition low-resonance Rayleigh scattering signal with buffer solution
Line 0mL)；After chitosan is added in brilliant blue-buffer solution system (under acid condition), generating a new tool, there are two apparent
The Resonance Rayleigh Scattering Spectra of scattering peak (345nm, 445nm), in terms of spectrum, Resonance Rayleigh Scattering intensity is dense with chitosan
The increase of degree and enhance, and it is in a linear relationship in certain mass concentration range；Spectrally there are two peaks, and each peak value is in line
Property be incremented by, but R can be made using dual wavelength2More preferably, therefore this paper experimental results are that dual wavelength measures.
2. influences of the pH of different buffer solutions and buffer solution for system Resonance Rayleigh Scattering intensity
First pH2-6 ranges measure system Δ I, as can be seen from Figure 2, there is the trend of falling after rising in Δ I, 2.5-3.5 it
Between have peak value, it is thus determined that pH value be 3.0.
B-R buffer solutions, HAc-NaAc buffer solutions, glycine-HCI buffer solution, citric acid-phosphorus have been tested respectively
Influence of the sour disodium hydrogen buffer solution to brilliant blue-chitosan system.As a result such as Fig. 3, the results showed that in citrate-phosphate disodium hydrogen
Intensity higher in buffer solution, therefore the acidity of the citrate-phosphate disodium hydrogen buffer solution control system of pH3.0 is selected, most preferably
Dosage is 1.0mL.
3. the influence of brilliant blue concentration and its addition for system Resonance Rayleigh Scattering intensity
Having investigated brilliant blue concentration and its addition influences system, sets brilliant blue a concentration of 2 × 10-4mol/mL；Addition
(mL) it is：0.5,1,1.5,2,2.5,3,3.5,4, as a result such as Fig. 4, as a result show that Δ I constantly rises, therefore consider to increase brilliant blue
A concentration of 8 × 10-4Mol/mL, addition (mL) are：0.5,1,1.5,2,2.5,3, Δ I has peak when being as a result shown in 1.5mL
Value, when brilliant blue a concentration of 1 × 10-3Mol/L, the Δ I of system reaches maximum when addition is 1mL, therefore selects 1mL1.0 × 10-3Mol/L brilliant blue solution, to obtain higher sensitivity.
Influence of the buffer solution to brilliant blue-chitosan system.The result shows that in citrate-phosphate disodium hydrogen buffer solution
Intensity higher, therefore the acidity of the citrate-phosphate disodium hydrogen buffer solution control system of pH3.0 is selected, optimum amount is
1.0mL。
4. influence of the reagent addition sequence for system Resonance Rayleigh Scattering intensity
Influence of 3 kinds of different reagent addition sequences to reaction system, experimental result such as Fig. 5 are tested, optimal sequence is
Chitosan-citrate-phosphate disodium hydrogen buffer solution-brilliant blue solution, but since it is linear poor, and citrate-phosphate disodium hydrogen
The addition sequence of buffer solution-brilliant blue solution-chitosan and chitosan-citrate-phosphate disodium hydrogen buffer solution-brilliant blue solution
Addition sequence Δ I be not much different, and the former is more excellent for linear ratio, therefore selected addition sequence is that citrate-phosphate disodium hydrogen delays
Rush solution-brilliant blue solution-chitosan.
5. influence of the reaction temperature for system Resonance Rayleigh Scattering intensity
Its influence to system is investigated at different temperatures, during the experiment, the system after heating has no color change,
Resonance Rayleigh Scattering intensity as shown in fig. 6, when 40 DEG C Δ I highests, however at 40 DEG C, the linear relationship of standard curve compared with
It is not much different under difference, slope and room temperature, therefore the selected measurement at normal temperatures of system.
6. influence of the stabilization time for system Resonance Rayleigh Scattering intensity
At ambient temperature, the stability of system 240min Nei has been inquired into, as a result such as Fig. 7, the results showed that brilliant blue solution
Δ I is opposite stable in 45min with chitosan reaction, and 45min starts rear Δ I and drastically downward trend is presented.Therefore, experiment is answered
It measures and finishes in 45min.
7. the influence of ionic strength
By preparing different NaCl solutions, in addition system, the ionic strength that observation system allows, as a result such as Fig. 8, from
Fig. 8 can be seen that chitosan-brilliant blue system when ionic strength is less than 0.01mol/L, and system is relatively stable；It is more than
When 0.01mol/L, concentration is higher, and influence is bigger.
8. interference--free experiments
Disturbed condition of 20 kinds of substances to this method is investigated, when chitosan concentration is 1 μ g/mL, relative error≤± 5%
When, the permission multiple of coexisting substances is shown in Table 1.
The influence of 1 coexisting substances of table
As seen from Table 1, iron, copper, calcium, aluminium influence big on reminding, and need to be sheltered with screening agent, 10 times, aluminium can use
0.01mol/LEDTA screening agents are sheltered, and 10 times of calcium ions then use 1mL100 μ g/mL and 0.4% tartaric acid to shelter, and are measuring sample
Two kinds of screening agents are added before product and reduce the error generated in its continuous mode.
9. influence of the different deacetylations to system
Under the best experimental conditions, measurement result difference of the system to the chitosan of different deacetylations is investigated.Point
It is the chitosan that 85% and 90% its molecular weight is 60 not investigated deacetylation.As a result such as Fig. 9, by statistical analysis, no
For chitosan result with deacetylation there is no the significance difference opposite sex, i.e., the chitosan of different deacetylations does not influence system.
The present invention has following technical advantage compared with prior art：
1) good linearity, detection limit are low：Under optimum experimental condition, according to the chitosan pair of determination of experimental method various concentration
The Δ I answered draws standard curve, the results showed that, chitosan exists within the scope of concentration 0.005-1.8 μ g/mL L with Δ I good
Linear relationship, the range of linearity is wide, detection be limited to 5.48 × 10-2μg/mL。
2) a kind of method that dual wavelength resonance rayleigh light scattering method measures chitosan content is established, this method measures poly- by shell
The influence of the molecular weight of sugar, in actual sample measurement, the standard items that need to be considered as close molecular weight do quantitative criterion, have
Reagent is cheap, high sensitivity, strong antijamming capability, favorable reproducibility and it is easy to operate the advantages that.
Description of the drawings
The Resonance Rayleigh Scattering collection of illustrative plates of Fig. 1 brilliant blues-chitosan system.
Influence diagram of Fig. 2 pH value of buffer solution to system Resonance Rayleigh Scattering intensity.
Influence diagram of Fig. 3 differences buffer solution to system Resonance Rayleigh Scattering intensity.
Influence diagram of Fig. 4 brilliant blues addition to system Resonance Rayleigh Scattering intensity.
Fig. 5 reagents addition sequence is to system Resonance Rayleigh Scattering intensity effect figure.
Fig. 6 reaction temperatures are to system Resonance Rayleigh Scattering intensity effect figure.
Fig. 7 stabilization times are to system Resonance Rayleigh Scattering intensity effect figure.
Fig. 8 ionic strengths are to system Resonance Rayleigh Scattering intensity effect figure.
Fig. 9 deacetylating degree of chitosan is to system Resonance Rayleigh Scattering intensity effect figure.
Specific implementation mode
The present invention is further described below by way of specific embodiment, but the invention does not limit the present invention in any way
The range of patent protection.
Embodiment
A kind of method that dual wavelength resonance rayleigh light scattering method measures chitosan content comprising following step：
1) standard curve of the chitosan concentration of Δ I and different molecular weight is drawn
The chitosan standard solution of 1.0mL a certain concentration gradients, 1.0mLpH=3.0 lemons are added into 10mL colorimetric cylinders
Acid-disodium hydrogen phosphate buffer solution, 1.0mL a concentration of 1.0 × 10-3The brilliant blue solution of mol/L, is settled to using distilled water three times
Scale, and fully shake up, room temperature 10min is placed it in, takes 1ml mixed solutions that quartz colorimetric utensil is added, in F-2500 type fluorescence
On spectrophotometer, with λex=λemScanning is synchronized, records each detection architecture of chitosan-containing respectively in maximum resonance Rayleigh
The resonance scattering intensity value I of system at dispersion wavelength λ=345nm1, wherein reagent blank system at λ=345nm resonance dissipate
Penetrate intensity value I01, and calculate Δ I1=I1-I01；Each detection architecture of chitosan-containing is recorded in secondary big Resonance Rayleigh Scattering wave simultaneously
Resonance Rayleigh Scattering intensity I at long λ=445nm2With the Resonance Rayleigh Scattering intensity I of blank reagent02, calculate Resonance Rayleigh Scattering
Strength difference Δ I2=I2-I02, wherein Δ I=Δs I1+ΔI2.Establish the standard curve C1 of Δ I and low molecular chitosan concentration；
Low molecular chitosan solution is replaced using middle-molecular-weihydroxyethyl chitosan solution, Δ I is established and the standard of middle molecular chitosan concentration is bent
Line C2；Low molecular chitosan solution is replaced using high molecular weight chitosan solution, establishes the mark of Δ I and polymer chitosan concentration
Directrix curve C3.
The chitosan result of 2 different molecular weight of table
2) preparation of the sample working solution of 10 μ g/mL：Chitosan capsules are gone into capsule shells, weigh 0.04g in 100mL capacity
It in bottle, is dissolved with 0.5mol/L glacial acetic acid, constant volume obtains stock sample solution.Storing solution, filtrate warp are filtered with funnel absorbent cotton
6000r/min, centrifuge 20min, take supernatant 2.5mL in 100mL volumetric flasks, and constant volume obtains the sample of a concentration of 10 μ g/mL
Product working solution.
3) according to chitosan molecule amount selection criteria curve：Δ I is drawn using detection method in step 1) and sample works
The standard curve of liquid, and by it compared with the chitosan standard curve of different molecular weight, according to the Δ I and sample working solution of sample
Standard curve regression equation determine the chitosan in sample molecular weight size, according to the molecule of corresponding chitosan
The size of amount determines used regression equation；
With sample working solution, empirically method draws sample curves, compared with the chitosan standard curve of different molecular weight,
As a result the equation of linear regression for being sample is Δ I=2014.20c+30.05, related coefficient 0.9958, as a result with middle-molecular-weihydroxyethyl
Chitosan standard curve is close, using the standard curve of middle-molecular-weihydroxyethyl chitosan as quantitation curves.
4) chitosan content determines in sample：Sample working solution 1mL is taken, is measured by step 1) detection method, in wave
Its Resonance Rayleigh Scattering strength difference Δ I is measured at long 345nm and 445nm1With Δ I2, and calculate Δ I=Δs I1+ΔI2, by Δ
I substitutes into equation of linear regression and acquires the content of chitosan in sample, while doing mark-on reclaims experiment.
Sample working solution 1mL is taken, empirically method is measured, its Rayleigh that resonates is measured at wavelength 345nm and 445nm
Scattering strength difference DELTA I1With Δ I2, and calculate Δ I=Δs I1+ΔI2, Δ I is substituted into equation of linear regression and acquires shell in sample
The content of glycan, while doing mark-on reclaims experiment.As a result it is：The content of chitosan is 251.4mg/g in Hypon chitosan capsules,
RSD is 1.1%.Recovery of standard addition is shown in Table 3.
The recovery of standard addition of 3 chitosan of table
In an experiment, the citrate-phosphate disodium hydrogen buffering that 1.0mLpH=3.0 is sequentially added in the colorimetric cylinder of 10mL is molten
Liquid, 1.0mL a concentration of 1.0 × 10-3The brilliant blue solution of mol/L, the chitosan solution of a concentration of 10 μ g/mL of 1.0mL, uses distilled water
It is settled to scale, and 10min is stored at room temperature after fully shaking up, takes 1ml mixed solutions that quartz colorimetric utensil is added, it is glimmering in F-2500 types
On light spectrophotometer, with λex=λemScanning is synchronized, is completed in being measured in 45min.Each inspection of chitosan-containing is recorded respectively
The resonance scattering intensity value I of survey system system at maximum resonance Rayleigh scattering wavelength X=345nm1, wherein reagent blank is in λ
The resonance scattering intensity value I of system at=345nm01, and calculate Δ I1=I1-I01；Each detection body of chitosan-containing is recorded simultaneously
Tie up to Resonance Rayleigh Scattering intensity I at time big Resonance Rayleigh Scattering wavelength X=445nm2With the Resonance Rayleigh Scattering of blank reagent
Intensity I02, calculate Resonance Rayleigh Scattering strength difference Δ I2=I2-I02, wherein Δ I=Δs I1+ΔI2..Chitosan is in concentration
With Δ I there are good linear relationship within the scope of 0.005-1.8 μ g/mL, the range of linearity is wide, and detection is limited to 5.48 × 10-2μg/
mL.The present invention establishes a kind of method that dual wavelength resonance rayleigh light scattering method measures chitosan content, and this method measures poly- by shell
The influence of the molecular weight of sugar, in actual sample measurement, the standard items that need to be considered as close molecular weight do quantitative criterion, have
Reagent is cheap, high sensitivity, strong antijamming capability, favorable reproducibility and it is easy to operate the advantages that.
The range of linearity and detection limit
Under optimum experimental condition, according to the corresponding Δ I of the chitosan of determination of experimental method various concentration, with Δ I to shell
Glycan concentration drawing curve, chitosan mass concentration are in good linear pass with Δ I within the scope of 0.005-1.8 μ g/mL
System, equation of linear regression y=2014.20x+30.05, coefficient R2It is 0.9958.Empirically method parallel determination blank
It is measured each 13 times with minimum pipe, acquiring detection according to CL=3s/k (s is standard deviation, and k is the slope of regression equation) is limited to
5.48×10-2μg/mL。
Claims (6)
1. a kind of method that dual wavelength resonance rayleigh light scattering method measures chitosan content comprising following step：
1) standard curve of the chitosan concentration of Δ I and different molecular weight is drawn：It is centainly dense that 1.0mL is added into 10mL colorimetric cylinders
Spend the chitosan standard solution of gradient, 1.0mL buffer solutions, 1.0mL a concentration of 1.0 × 10-3The brilliant blue solution of mol/L uses
Distilled water is settled to scale three times, and fully shakes up, and places it in room temperature 10min, takes 1ml mixed solutions that quartz cuvette is added
Ware, on F-2500 type sepectrophotofluorometers, with λex=λemScanning is synchronized, records each detection of chitosan-containing respectively
The resonance scattering intensity value I of system system at maximum resonance Rayleigh scattering wavelength X=345nm1, wherein reagent blank λ=
The resonance scattering intensity value I of system at 345nm01, and calculate Δ I1=I1-I01；Each detection architecture of chitosan-containing is recorded simultaneously
The Resonance Rayleigh Scattering intensity I at secondary big Resonance Rayleigh Scattering wavelength X=445nm2It is strong with the Resonance Rayleigh Scattering of blank reagent
Spend I02, calculate Resonance Rayleigh Scattering strength difference Δ I2=I2-I02, wherein Δ I=Δs I1+ΔI2；Establish Δ I and low molecule shell
The standard curve C1 of Glycan concentration；Low molecular chitosan solution is replaced using middle-molecular-weihydroxyethyl chitosan solution, Δ I is established and divides in
The standard curve C2 of seed chitosan concentration；Using high molecular weight chitosan solution replace low molecular chitosan solution, establish Δ I with
The standard curve C3 of polymer chitosan concentration；
2) preparation of the sample working solution of 10 μ g/mL：Chitosan capsules are gone into capsule shells, weigh 0.04g in 100mL volumetric flasks
In, it is dissolved with 0.5mol/L glacial acetic acid, constant volume obtains stock sample solution.Storing solution is filtered with funnel absorbent cotton, filtrate is through 6000r/
Min, centrifuge 20min, take supernatant 2.5mL in 100mL volumetric flasks, and constant volume obtains the sample work of a concentration of 10 μ g/mL
Liquid.
3) according to chitosan molecule amount selection criteria curve：Δ I and sample working solution are drawn using detection method in step 1)
Standard curve, and by it compared with the chitosan standard curve of different molecular weight, according to the mark of the Δ I and sample working solution of sample
The regression equation of directrix curve determines the size of the molecular weight of the chitosan in sample, according to the molecular weight of corresponding chitosan
Size determines used regression equation；
4) chitosan content determines in sample：Sample working solution 1mL is taken, is measured by step 1) detection method, in wavelength
Its Resonance Rayleigh Scattering strength difference Δ I is measured at 345nm and 445nm1With Δ I2, and calculate Δ I=Δs I1+ΔI2, by Δ I
It substitutes into equation of linear regression and acquires the content of chitosan in sample, while doing mark-on reclaims experiment.
2. the method that a kind of dual wavelength resonance rayleigh light scattering method according to claim 1 measures chitosan content, feature
It is, the buffer solution is B-R buffer solutions, HAc-NaAc buffer solutions, glycine-HCI buffer solution, citrate-phosphate
One kind in disodium hydrogen buffer solution.
3. the method that a kind of dual wavelength resonance rayleigh light scattering method according to claim 2 measures chitosan content, feature
It is, the buffer solution is citrate-phosphate disodium hydrogen buffer solution.
4. the method that a kind of dual wavelength resonance rayleigh light scattering method according to claim 3 measures chitosan content, feature
It is, the pH of the citric acid-sodium citrate buffer is 3.0.
5. the method that a kind of dual wavelength resonance rayleigh light scattering method according to claim 1 measures chitosan content, feature
It is, the addition sequence of reagent is to be firstly added citrate-phosphate disodium hydrogen buffer solution in step 1, and it is molten that brilliant blue is secondly added
Liquid is added lauryl sodium sulfate anionic surfactant solution, is eventually adding chitosan solution again.
6. the method that a kind of dual wavelength resonance rayleigh light scattering method according to claim 1 measures chitosan content, feature
It is, the concentration range of chitosan is 0.005-1.8 μ g/mL in step 1).
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| CN110749574B (en) * | 2019-11-05 | 2021-11-02 | 广东药科大学 | Method for measuring perfluorooctane sulfonate by dual-wavelength resonance Rayleigh scattering method and application |
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