CN108318455A - A method of chitosan content is measured by the resonance rayleigh light scattering method of OP emulsifier enhanced sensitivities - Google Patents
A method of chitosan content is measured by the resonance rayleigh light scattering method of OP emulsifier enhanced sensitivities Download PDFInfo
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Abstract
The present invention relates to a kind of methods that the resonance rayleigh light scattering method by OP emulsifier enhanced sensitivities measures chitosan content, belong to big Molecular Detection field.The present invention utilizes under certain condition, after OP sensitizers intervene Congo red and chitosan system, its Resonance Rayleigh Scattering Spectra intensity increases severely, and within the scope of a certain concentration, Resonance Rayleigh Scattering Spectra enhancement value is directly proportional to the concentration of chitosan, in this, as the quantitative basis of chitosan, the resonance rayleigh light scattering method for measuring chitosan is established.It is experimentally confirmed, this method is easy to operate, accuracy is high, stability is good, is suitble to the measurement of chitosan content in chitosan raw material and compound sample.
Description
Technical field
The present invention relates to a kind of methods that the resonance rayleigh light scattering method by OP emulsifier enhanced sensitivities measures chitosan content, belong to
In big Molecular Detection field.
Background technology
Chitosan (chitosan, CTS) is widely present in unicellular lower eukaryote mushroom, the cell of algae, section branch animal shrimp, crab,
It is the second largest biological polyoses for being only second to cellulose on the earth in the shell of insect, is unique alkali containing free amine group in nature
Property cation edibility animal origin.The excellent characteristic of chitosan makes it in the side such as medicine, health food, weaving and environmental protection
Face all has extensive purposes, and the application especially in terms of health food is increasingly extensive.With the main difference of other food fibres
Place is that it is a kind of unique wholefood fiber of cationic.Major function includes absorbing fats acid, cholesterol etc.,
Absorption of human body is reduced, while can also slow down hypertension and diabetic symptom, increases cellular immune function, therefore become healthy food
The favorite of industry.
Chitosan content assay method have very much, as spectrophotometry, fluorescence method, chromatography, resonance rayleigh light scattering method and
Electrochemical process etc..The method that resonance rayleigh light scattering method measures chitosan has the remarkable advantages such as high sensitivity, easy to operate, herein
Using OP emulsifier enhanced sensitivities resonance rayleigh light scattering method measure chitosan content method there is not been reported.
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 the resonance rayleigh light scattering method by OP emulsifier enhanced sensitivities measures chitosan content, the present invention
After intervening Congo red and chitosan system using OP sensitizers under certain condition, Resonance Rayleigh Scattering Spectra intensity increases severely, and
Within the scope of a certain concentration, Resonance Rayleigh Scattering Spectra enhancement value is directly proportional to the concentration of chitosan, in this, as chitosan
Quantitative basis establishes the resonance rayleigh light scattering method for measuring chitosan.It is experimentally confirmed, this method is easy to operate, accuracy
Height, stability are good, are suitble to the measurement of chitosan content in chitosan raw material and compound sample.
A method of chitosan content, packet are measured by the resonance rayleigh light scattering method of OP emulsifier enhanced sensitivities
Include following step：
1) standard curve of the chitosan of Δ I and various concentration is drawn
The chitosan standard solution of addition 0.5mL a certain concentration gradients into 10mL colorimetric cylinders, the buffer solution of 1.5mL,
The OP emulsifiers and 0.5mL a concentration of 1 × 10 of a concentration of 7.6 × 10-3mol/L of 1.5mL-3The Congo red solution of mol/L,
Using fully being shaken up after distilled water constant volume, take 1ml mixed solutions that quartz colorimetric utensil is added after placing it in room temperature 10min, in F-
On 2500 type sepectrophotofluorometers, with λex=λemScanning is synchronized, records each detection architecture of chitosan-containing respectively most
The resonance scattering intensity value I of system, wherein reagent blank body at λ=376nm at big Resonance Rayleigh Scattering wavelength X=376nm
The resonance scattering intensity value I of system0, and calculate Δ I=I-I0, establish the standard curve C of Δ I and chitosan concentration；
2) preparation of the sample working solution of 10 μ g/mL：Chitosan capsules are gone into capsule shells, 0.04g is weighed and holds in 100mL
It in measuring 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) chitosan content determines in sample：Take sample working solution 1mL, be measured by step 1) detection method, λ=
Its Resonance Rayleigh Scattering intensity value I is measured at 376nm, and calculates Δ I=I-I0, Δ I is substituted into equation of linear regression and acquires sample
The content of chitosan in product, while doing mark-on reclaims experiment.
It is described slow as described above by the method that the resonance rayleigh light scattering method of OP emulsifier enhanced sensitivities measures chitosan content
Fliud flushing is B-R buffer solutions, glycine-hydrochloride buffer, phthalic acid-hydrochloride buffer, disodium hydrogen phosphate-citric acid
One kind in buffer solution；Applicants experimentally found that in disodium hydrogen phosphate-citrate buffer solution, chitosan-is Congo red
The Resonance Rayleigh Scattering intensity of ionic associate is larger and system is more stable.In addition it has been found that system is in pH=2.5 phosphorus
Sour disodium hydrogen-citrate buffer solution medium sensitivity highest, scattering strength is larger and stablizes, and therefore, selects the phosphoric acid of pH=2.5
Disodium hydrogen-citrate buffer solution is as buffer solution.
As described above by the method that the resonance rayleigh light scattering method of OP emulsifier enhanced sensitivities measures chitosan content, step 1
The addition sequence of middle reagent is to be firstly added Congo red solution, and OP emulsifiers are secondly added, and disodium hydrogen phosphate-lemon is added again
Lemon acid buffer, is eventually adding chitosan solution.
As described above by the method that the resonance rayleigh light scattering method of OP emulsifier enhanced sensitivities measures chitosan content, step 1)
0.05~1.0 μ g/mL of concentration range of middle chitosan.
Sample pre-treatments：Chitosan capsules are gone into capsule shells, 0.04g is weighed in 100mL volumetric flasks, uses 0.5mol/L
Glacial acetic acid dissolves, and constant volume obtains stock sample solution.Filter storing solution with funnel absorbent cotton, filtrate through 6000r/min, centrifuge from
Heart 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, empirically method is measured, its Resonance Rayleigh Scattering intensity value is measured at 376nm
I, and calculate Δ I=I-I0, Δ I is substituted into equation of linear regression and acquires the content of chitosan in sample, while doing mark-on reclaims
Experiment.
Test example：
1. the Resonance Rayleigh Scattering collection of illustrative plates of Congo red-OP- chitosans
In a series of 10mL colorimetric cylinders, it is sequentially separately added into Congo red operation liquid 0.5mL, op sensitizer
The disodium hydrogen phosphate of 1.0mL, pH=2.5-citric acid solution 1.5mL, chitosan operate liquid 0,0.05,0.1,0.2,
0.4,1.0mL is diluted to graduation mark with distilled water, shakes up.Make reference with reagent blank, scans Resonance Rayleigh Scattering Spectra, show
In Fig. 1.CTS concentration (mgL from bottom to up in Fig. 1- 1)：0,0.05,0.1,0.2,0.4,1.0.
The result shows that Resonance Rayleigh Scattering intensity is relatively low when simple Congo red, combined with CTS and in the sensitization of OP
Under, Resonance Rayleigh Scattering intensity significantly increases, be in wavelength there is peak value at 376nm, and Resonance Rayleigh Scattering intensity with
It the content increase that CTS is added and increases.
2. influence of the sensitizer for system Resonance Rayleigh Scattering intensity
Two groups of colorimetric cylinders, a group reagent blank, another group of addition 2mL chitosan standard are taken to use solution, be separately added into pH
For 2.5 B-R buffer solutions 2.0mL, 1.0 × 10-3The Congo red solution 1.0mL of mol/L.Using reagent blank as reference, examine respectively
Examine hexadecyl (9.12 × 10-4Mol/L), lauryl sodium sulfate (0.14mol/L), NaLS
(0.086mol/L), polysorbas20 (0.6g/L), Tween 80 (0.14g/L), OP emulsifiers (1.9 × 10-3) and polyethylene mol/L
Influence of the alcohol (1%) to reaction system scattering strength.The result shows that：OP emulsifiers are to the Congo red ionic associate of chitosan-
RRS intensity increases larger and stablizes.
3. influences of the pH of buffer solution for system Resonance Rayleigh Scattering intensity
Two groups of colorimetric cylinders, a group reagent blank, another group of addition 2mL chitosan standard are taken to use solution, be separately added into not
With the B-R buffer solutions 2.0mL of pH value, 1.0 × 10-3Mol/L Congo red solution 1.0mL, OP emulsifier solutions 2mL.With reagent
Blank is reference, measures the RRS intensity I of corresponding pH valueRRS.Measurement result is shown in Fig. 1 and Fig. 2.From Figure 2 it can be seen that pH 2.4~2.6
Between scattering strength it is larger and stablize.Therefore, buffer system selects pH2.5.
4. influence of the different buffer solutions for system Resonance Rayleigh Scattering intensity
Investigate B-R buffer solutions, glycine-hydrochloride buffer, phthalic acid-hydrochloric acid respectively under the acidity of pH2.5
The influence of buffer solution, disodium hydrogen phosphate-citrate buffer solution to reaction system scattering strength, the results showed that：In phosphoric acid hydrogen two
In sodium-citrate buffer solution, the RRS intensity of the Congo red ionic associate of chitosan-is larger and system is more stable.
5. disodium hydrogen phosphate-influence of the citrate buffer solution addition for system Resonance Rayleigh Scattering intensity
Two groups of colorimetric cylinders are taken, a group reagent blank, another group adds 2.0mL chitosan standards to use solution, is separately added into 1.0
×10-3The Congo red solution 1.0mL of mol/L, fixed disodium hydrogen phosphate-citrate buffer solution pH2.5, OP emulsifier solution 2mL,
Change buffer solution dosage.Using reagent blank as reference, the scattering strength of corresponding buffer solution addition is measured, measurement result is shown in
Fig. 3.As seen from Figure 3, solution is when buffer solution addition is 1.5mL, Δ IRRSHighest.So selection optimized buffer solution adds
It is 1.5mL to enter amount.
6. influence of the Congo red addition for system Resonance Rayleigh Scattering intensity
Two groups of colorimetric cylinders are taken, a group reagent blank, another group adds 2.0mL chitosan standards to use solution, fixed phosphoric acid hydrogen
Disodium-citric acid solution pH2.5, dosage 1.5mL, OP emulsifier solution 2mL change Congo red operation liquid dosage.With
Reagent blank is reference, the scattering strength of the corresponding Congo red addition of measurement, such as Fig. 4, the experimental results showed that, when Congo red addition
When amount is 0.5mL, Δ IRRSHighest.So selecting that the amount of Congo red operation liquid is added to be 0.5mL.
Influence of the 7.OP emulsifiers addition for system Resonance Rayleigh Scattering intensity
When the concentration of OP emulsifiers reaches critical micelle concentration, OP molecules will be assembled inside solution, can be formed
Micella wraps up associated matter, so that complex volume is increased, to reach effect of enhanced sensitivity, on this basis, has prepared 7.6 × 10- 3The OP emulsifier solutions of mol/L carry out addition experiment.
Two groups of colorimetric cylinders are taken, a group reagent blank, another group adds 2.0mL chitosan standards to use solution, fixed phosphoric acid hydrogen
Disodium-citric acid solution pH2.5, dosage 1.5mL, Congo red operation liquid 2mL, change OP enhanced sensitivities agent solution (7.6 ×
10-3Mol/L dosage).Using reagent blank as reference, the scattering strength of corresponding OP enhanced sensitivities agent solution addition, such as Fig. 5 are measured.
The experimental results showed that when OP enhanced sensitivity agent solution additions are 1.0mL, Δ IRRSHighest and gradually it is intended to steady.So selection
The amount that OP enhanced sensitivity agent solutions are added is 1.0mL.
8. influence of the reaction temperature for system Resonance Rayleigh Scattering intensity
Under more excellent experiment condition, take 2 groups of 10mL colorimetric cylinders (standard pipe and reagent blank pipe), standard pipe that CTS marks are added
Quasi- solution 2.00mL, then heating water bath at a temperature of 20 DEG C, 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C, measures and places different temperatures
Δ I under systemRRS.With Δ IRRS- T maps, and refers to figure below.Such as Fig. 6, the scattering strength value substantially constant in 40 DEG C, with temperature
The increase of degree, Δ IRRSIt is slightly decreased.So experimental selection carries out at room temperature.
9. influence of the stabilization time for system Resonance Rayleigh Scattering intensity
Under more excellent experiment condition, take 2 groups of 10mL colorimetric cylinders (standard pipe and reagent blank pipe), standard pipe that CTS marks are added
Quasi- solution 2.00mL measures the Δ I of system after placing 5~240minRRS.With Δ IRRS- t maps, and refers to Fig. 7.As seen from Figure 7,
The scattering strength value substantially constant in 120min, with the increase of standing time, Δ IRRSIt is slightly decreased.
10. influence of the addition sequence for system Resonance Rayleigh Scattering intensity
Under optimum experimental condition, by chitosan, disodium hydrogen phosphate-citric acid solution, Congo red, OP sensitizers
It is added in a different order, shares 24 kinds of combinations；The result shows that Congo red and OP emulsifiers are first added, it is mixed to add buffer solution
It closes, is eventually adding the sequence of chitosan, RSS intensity highests.So poly- using Congo red-OP- buffer solutions-shell in subsequent experimental
The addition sequence of sugar.
11. influence of the ionic strength for system Resonance Rayleigh Scattering intensity
10mL colorimetric cylinders are taken, it is molten that Congo red operation liquid 0.5mL, op sensitizer 1.0mL, pH=2.5 buffering is added in blank group
Liquid 1.5mL；Experimental group sequentially adds Congo red operation liquid 0.5mL, OP sensitizer 1.0mL, pH=2.5 buffer solution 1.5mL,
1mL chitosan solutions (10 μ g/mL), 0,0.01,0.05,0.1,0.3,0.5,0.7,1.0,3.0mol/L NaCl solutions
1.0ml adds distilled water to scale, mixing.The resonance scattering spectroscopy for measuring solution under different ions concentration, is as a result shown in Fig. 8.
As shown in Figure 8, the reaction with the NaCl general trends changed be first it is constant, after be gradually reduced, in 0-
Interior reaction tends to be steady when 0.001mol/L, thus it is speculated that may be that NaCl concentration is relatively low, is not impacted to reaction system.
0.005mol/L increases Δ I with ionic strengthRRSDecline, is in negatively influencing, reason may be that NaCl destroys the Congo red ions of CTS-
The hydrophobic structure of associated matter so that Δ IRRSDecline.Therefore the NaCl that measurement actual sample process preferably controls sample solution is dense
Degree is no more than 0.005mol/L, could make that measurement result is accurate and reliable in this way.
12. influence of the chitosan of different deacetylations and molecular weight for system Resonance Rayleigh Scattering intensity
In disodium hydrogen phosphate-citric acid buffer system, six groups of colorimetric cylinders are taken, draw four kinds of different molecular weights respectively
The different deacetylation 60cps 85% of 45cps 85%, 60cps 85%, 95cps 85%, 188cps 85% and three kind,
The standard curve of the chitosan of 60cps 90%, 60cps 95%.Six groups of chitosan standard solution mass concentrations (0.05,0.1,
0.2,0.4,0.8,1.0 μ g/mL), each parallel 3 parts, using reagent blank as reference, the resonance for measuring corresponding molecular weight chitosan is auspicious
Sharp scattering strength.
As a result, it has been found that it is identical in mass concentration, in the case where chitosan is all 85% deacetylation, different molecular
Amount is the Resonance Rayleigh Scattering intensity linear equation of the chitosan solution of 45cps, 60cps, 95cps, 188cps, such as Fig. 9, line
Property equation is respectively y=1331.5x-68.727, R2=0.9979；Y=1118.1x- 12.634, R2=0.9966；Y=
1333.2x-51.336 R2=0.9972；Y=1205.3x+48.443, R2=0.9904.Through slope significance test, P=
0.947 (＞ 0.05), there was no significant difference.Measure chitosan content in this approach is not influenced by molecular size range.
Identical in mass concentration, chitosan is all under the molecular weight of 60cps, different deacetylations are 85%,
90%, the Resonance Rayleigh Scattering intensity linear equation of 95% three groups of chitosan solutions, such as Figure 10, linear equation is respectively y
=1118.1x-12.634, R2=0.9966；Y=1524.8x-29.759, R2=0.9975；Y=954.35x+5.8069, R2
=0.9972.Through slope significance test, P=0.744 (＞ 0.05) is not present significant difference, measures shell in this approach
Glycan content is not also influenced by deacetylation.
13. influence of the influence of coexisting substances for system Resonance Rayleigh Scattering intensity
Currently, such as crust cellulose capsule of the common health products containing CTS in the market, major auxiliary burden ingredient have gelatin, starch,
Dextrin etc..In the solution of a concentration of 1.0 μ g/mL of chitosan mass, possible coexisting substances in crust cellulose capsule are separately added into,
Interference test measurement is carried out, with not plus the standard solution measurement result of interfering substance compare, calculates about ± 5% relative error of generation
When the concentration of each coexisting substances is added, the results are shown in Table 1.The result shows that interfere measurement result larger substance, as zinc, iron from
Son, magnesium ion, calcium ion, copper ion etc., can be added EDTA or bad hematic acid adds tartaric acid to shelter.
Influence of 1 coexisting substances of table to reaction system
14. influence of the screening agent for system Resonance Rayleigh Scattering intensity
The presence of coexisting substances can cause prodigious error to the measurement of sample, so needing that a certain amount of screening agent is added
The coexisting ion that system is affected is sheltered.In the solution of a concentration of 1.0 μ g/mL of chitosan mass, respectively
10 μ g/mL coexisting substances ions are not added, add screening agent, and not plus the standard solution measurement result of screening agent compare, calculating
The concentration of each screening agent is added when generating about ± 5% relative error, the results are shown in Table 2.
Influence of 2 screening agent of table to coexisting ion
As seen from table, it finally determines and 1.5mL EDTA (0.01moL/L) is added in system, deposited in 25mg/L bad hematic acid
Under, 4% tartaric acid of 1mL can effectively shelter coexisting ion.It has been carried out at the same time the experiment that screening agent separately exists in system,
Screening agent works well there is no being had an impact to the Resonance Rayleigh Scattering intensity of original 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, there are good with Δ I within the scope of 0.05~1.0 μ g/mL of concentration for chitosan
Linear relationship, the range of linearity is wide, and detection is limited to 6.99ng/mL.
2) this method measurement is not influenced by chitosan molecule amount size and deacetylation, is acted on and being stablized under room temperature, and
This method is easy to operate, accuracy is high, stability is good, is suitble to the measurement of chitosan content in chitosan raw material and compound sample.
Description of the drawings
The Resonance Rayleigh Scattering collection of illustrative plates of the Congo red-OP- chitosans of Fig. 1.
Influence diagrams of the pH of Fig. 2 buffer solutions to system Resonance Rayleigh Scattering intensity.
Influence diagram of Fig. 3 disodium hydrogen phosphates-citrate buffer solution addition to system Resonance Rayleigh Scattering intensity.
The Congo red additions of Fig. 4 are to system Resonance Rayleigh Scattering intensity effect figure.
Fig. 5 OP emulsifiers additions are to system Resonance Rayleigh Scattering intensity effect figure.
Fig. 6 temperature is 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 chitosan molecules amount is to system Resonance Rayleigh Scattering intensity effect figure.
Figure 10 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 method of chitosan content being measured by the resonance rayleigh light scattering method of OP emulsifier enhanced sensitivities comprising Xia Shubu
Suddenly：
1) standard curve of the chitosan of Δ I and various concentration is drawn
The chitosan standard solution of 0.5mL a certain concentration gradients, the phosphorus of 1.5mLpH=2.5 are added into 10mL colorimetric cylinders
Sour disodium hydrogen-citrate buffer solution, the OP emulsifiers and 0.5mL a concentration of 1 of a concentration of 7.6 × 10-3mol/L of 1.5mL
×10-3The Congo red solution of mol/L takes 1ml to mix using fully being shaken up after distilled water constant volume after placing it in room temperature 10min
Quartz colorimetric utensil is added in solution, on F-2500 type sepectrophotofluorometers, with λex=λemScanning is synchronized, is recorded respectively
The resonance scattering intensity value I of each detection architecture of chitosan-containing system at maximum resonance Rayleigh scattering wavelength X=376nm,
The resonance scattering intensity value I of middle reagent blank system at λ=376nm0, and calculate Δ I=I-I0, it is dense with chitosan to establish Δ I
The standard curve C of degree；
2) preparation of the sample working solution of 10 μ g/mL：Chitosan capsules are gone into capsule shells, 0.04g is weighed and holds in 100mL
It in measuring 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) chitosan content determines in sample：Take sample working solution 1mL, be measured by step 1) detection method, λ=
Its Resonance Rayleigh Scattering intensity value I is measured at 376nm, and calculates Δ I=I-I0, Δ I is substituted into equation of linear regression and acquires sample
The content of chitosan in product, while doing mark-on reclaims experiment.
Sample working solution 1mL is taken, empirically method is measured, its scattering value I is measured at 470nm, and calculate Δ I=
I–I0, Δ I is substituted into equation of linear regression and acquires the content of chitosan in sample, while doing mark-on reclaims experiment.As a result it is：Sample
CTS average contents are 958.79mg/g, RSD 1.30% in product Ao Liwei chitosan capsules；CTS is flat in Hypon chitosan capsules
Equal content is 880.76mg/g, RSD 2.05%.Recovery of standard addition is shown in Table 3.
3 sample analysis result of table
In an experiment, 0.5mL a concentration of 1 × 10 is sequentially added in the colorimetric cylinder of 10mL-3The Congo red solution of mol/L,
The OP emulsifiers of a concentration of 7.6 × 10-3mol/L of 1.5mL, disodium hydrogen phosphate-citrate buffer solution of 1.5mLpH=2.5 and
A concentration of 10 μ g/mL chitosan solutions of 0.5mL, are settled to scale with distilled water, and be stored at room temperature 10min after fully shaking up, take
Quartz colorimetric utensil is added in 1ml mixed solutions, on F-2500 type sepectrophotofluorometers, with λex=λemScanning is synchronized, in
It measures and completes in 2h.Record the resonance scattering of system system at maximum resonance Rayleigh scattering wavelength X=376nm of chitosan-containing
The resonance scattering intensity I of intensity I, wherein the reagent blank system at λ=376nm0, and calculate Δ I=I-I0.Chitosan is dense
It spends within the scope of 0.05~1.0 μ g/mL with Δ I there are good linear relationship, the range of linearity is wide, and detection is limited to 6.99ng/mL.
This method measurement is not influenced by chitosan molecule amount size and deacetylation, is acted on and being stablized under room temperature, and this method operates
Simply, accuracy is high, stability is good, is suitble to the measurement of chitosan content in chitosan raw material and compound sample.
Linear relationship and detection limit
In a series of 10mL colorimetric cylinders, it is sequentially separately added into Congo red operation liquid 0.5mL, op sensitizer
The disodium hydrogen phosphate of 1.0mL, pH=2.5-citric acid solution 1.5mL, chitosan operate liquid 0,0.05,0.1,0.2,
0.4,1.0,2.0,3.0,4.0,5.0,6.0mL, is diluted to graduation mark with distilled water, shakes up.Make reference with reagent blank, measures
The resonance scattering spectroscopy of solution.The result shows that within the scope of 0.05~1.0 μ g/mL, resonance scattering intensity Δ I and chitosan are dense
It is in good linear relationship, regression equation Δ I=1505.1c+43.278, coefficient R=0.9982 to spend c.
According to experimental method, 0.05 μ g/mL of selection standard curve minimum, METHOD FOR CONTINUOUS DETERMINATION 13 times, while doing reagent blank
13 times, according to standard deviation and slope of standard curve, calculate this method detection be limited to 6.99ng/mL.
Claims (6)
1. a kind of method that resonance rayleigh light scattering method by OP emulsifier enhanced sensitivities measures chitosan content comprising Xia Shubu
Suddenly：
1) standard curve of the chitosan of Δ I and various concentration is drawn
The chitosan standard solution of 0.5mL a certain concentration gradients, the buffer solution of 1.5mL, 1.5mL are added into 10mL colorimetric cylinders
A concentration of 7.6 × 10-3The OP emulsifiers and 0.5mL a concentration of 1 × 10 of mol/L-3The Congo red solution of mol/L, uses steaming
It is fully shaken up after distilled water constant volume, takes 1ml mixed solutions that quartz colorimetric utensil is added after placing it in room temperature 10min, in F-2500 types
On sepectrophotofluorometer, with λex=λemScanning is synchronized, records each detection architecture of chitosan-containing respectively in maximum resonance
The resonance scattering intensity value I of system at Rayleigh scattering wavelength X=376nm, wherein the reagent blank system at λ=376nm are total to
Scattering strength of shaking value I0, and calculate Δ I=I-I0, establish the standard curve C of Δ I and 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) chitosan content determines in sample：Take sample working solution 1mL, be measured by step 1) detection method, λ=
Its resonance scattering intensity value I is measured at 376nm, and calculates Δ I=I-I0, Δ I is substituted into equation of linear regression, is acquired in sample
The content of chitosan, while doing mark-on reclaims experiment.
2. the resonance rayleigh light scattering method according to claim 1 by OP emulsifier enhanced sensitivities measures the side of chitosan content
Method, which is characterized in that the buffer solution be B-R buffer solutions, glycine-HCI buffer solution, phthalic acid-hydrochloride buffer,
One kind in disodium hydrogen phosphate-citrate buffer solution.
3. the resonance rayleigh light scattering method according to claim 2 by OP emulsifier enhanced sensitivities measures the side of chitosan content
Method, which is characterized in that the buffer solution is disodium hydrogen phosphate-citrate buffer solution.
4. the resonance rayleigh light scattering method according to claim 3 by OP emulsifier enhanced sensitivities measures the side of chitosan content
Method, which is characterized in that the pH of the disodium hydrogen phosphate-citrate buffer solution is 2.5.
5. the resonance rayleigh light scattering method according to claim 1 by OP emulsifier enhanced sensitivities measures the side of chitosan content
Method, which is characterized in that the addition sequence of solution is to be firstly added Congo red solution in step 1, OP emulsifiers is secondly added, again
Disodium hydrogen phosphate-citrate buffer solution is added, is eventually adding chitosan solution.
6. the resonance rayleigh light scattering method according to claim 1 by OP emulsifier enhanced sensitivities measures the side of chitosan content
Method, which is characterized in that 0.05~1.0 μ g/mL of concentration range of chitosan in step 1).
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