CN105486648B - A kind of resonance rayleigh light scattering method of quantitative analysis chitosan - Google Patents
A kind of resonance rayleigh light scattering method of quantitative analysis chitosan Download PDFInfo
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Abstract
The invention belongs to detection technique fields, specifically disclose a kind of resonance rayleigh light scattering method of quantitative analysis chitosan.For the present invention the study found that in faintly acid B R buffer systems, the ionic associate of Reactive Red 4 and chitosan generates strong Resonance Rayleigh Scattering characteristic peak, Resonance Rayleigh Scattering intensity and chitosan concentration at λ=342nmcIn good linear relationship, in the concentration range of 0.050~6.00 μ g/mL, linear equation is：ΔI=682.31c+ 114.95（c, μ g/mL）,R 2=0.9991,0.033 μ g/mL of detection limit.Accordingly, the resonance rayleigh light scattering method that chitosan is analyzed using Reactive Red 4 as probe quantitative is established, the rate of recovery of this method is 95.85~104.8%.
Description
Technical field
The present invention relates to detection technique fields, more particularly, to a kind of Resonance Rayleigh Scattering of quantitative analysis chitosan
Method.
Background technology
Chitosan（CTS）It is chitin deacetylation product, its chemical name is Chitosan (Isosorbide-5-Nitrae) -2- amino -2-
Deoxidation-D- glucans are the second largest natural organic polymer compounds that nature content is only second to cellulose.It is shell-fish
The main component of the ectoskeleton of (shrimp, crab) animal, insect.Chitosan and its derivative is important bioactive substance, has
Antitumor, anticoagulation, the functions such as Weight-reducing and lipid-lowering and strengthen immunity can be used as the addition of Medicines and Health Product, food, cosmetics etc.
Agent and the important source material for preparing artificial skin and operation suture thread etc..Therefore, chitosan is in biomedicine, environmental sanitation and food
Equal fields suffer from wide application prospect.
With chitosan extensive use, the accurate quantitative analysis of chitosan seems particularly significant.Currently, being used for chitosan both at home and abroad
The method of quantitative analysis is concentrated mainly on spectrophotometry and fluorescence method.Currently, spectrographic determination chitosan generally has ignored shell
Glycan is differed from thousands of to million as macromolecule natural products, molecular weight, when chitosan molecule amount and standard items in sample
When differing greatly, certain systematic error may be will produce, Accurate Determining is influenced.
Invention content
The present invention provides Reactive Red 4 in the spy as quantitative analysis chitosan to overcome the above-mentioned deficiency of the prior art
Application in needle.
It is a further object to provide a kind of using Reactive Red 4 as the method for the quantitative analysis chitosan of probe.
It is also another object of the present invention to provide a kind of resonance rayleigh light scattering methods of quantitative analysis chitosan.
To achieve the goals above, the present invention is achieved by following scheme：
The present invention has studied Reactive Red 4 and the spectral signature after chitosan association under weakly acidic condition first, and its poly- in shell
Application in sugared quantitative analysis.The result shows that in faintly acid B-R buffer systems, the ionic associate of Reactive Red 4 and chitosan
Strong Resonance Rayleigh Scattering characteristic peak, Resonance Rayleigh Scattering intensity and chitosan concentration are generated at λ=342nmcIn good line
Sexual intercourse, in the concentration range of 0.050~6.00 μ g/mL, linear equation is：ΔI = 682.31c+ 114.95（c, μ g/
mL）,R 2=0.9991,0.033 μ g/mL of detection limit.Accordingly, claimed Reactive Red 4 is as quantitative analysis chitosan
Probe in application.
In addition, the present invention also protect it is a kind of using Reactive Red 4 as the method for the quantitative analysis chitosan of probe.
A kind of resonance rayleigh light scattering method of quantitative analysis chitosan, includes the following steps：
The drafting of standard curve：N are configured at the chitosan solution of gradient concentration and 1 blank reagent, to n at gradient
It is separately added into the B-R buffer solutions of 2 times of volumes in the chitosan solution of concentration and 1 blank reagent, is then separately added into again
The Reactive Red 4 solution of volume, the solution to be measured of the chitosan at concentration gradient after being diluted after mixing are stood, detection
The Resonance Rayleigh Scattering intensity of chitosan solution to be measured specially synchronizes scanning with λ ex=λ em, records RRS spectrum, and
Measure ionic associate respectively at 342nmI RRSWith reagent blankI 0, standard curve is drawn, linear equation is further obtained：ΔI = 682.31c+ 114.95（c, μ g/mL）
In formulaΔI = I RRS-I 0；
Sample detection：Into the supernatant of the sample to be tested after preliminary treatment, the B-R bufferings that 2 times of volumes are added are molten
Then liquid adds isometric Reactive Red 4 solution, stands after mixing, Resonance Rayleigh Scattering intensity is detected, by above-mentioned line
Property equation calculation goes out the concentration of chitosan in sample to be tested.
Preferably, the time of the standing is within 120 minutes.
Preferably, a concentration of the 1.00 × 10 of the Reactive Red 4 solution-4The pH of mol/L, B-R buffer solution is 5.5.
Preferably, the step of sample to be tested preliminary treatment is as follows：The sample to be tested acetum of 0.5mol/L is dissolved,
It centrifuges until completely dissolved, obtains supernatant, detected for fluorescent quenching after supernatant dilution.
It is highly preferred that a kind of resonance rayleigh light scattering method of quantitative analysis chitosan, includes the following steps：
The drafting of standard curve：In the colorimetric cylinder of 10.00mL a series of, it is sequentially separately added into 1mL concentration point
Not Wei 0.5,1,3,5,10,20,30,40,50,60 μ g/mL chitosan solution, the B-R buffer solution buffer solutions of pH5.50
2.00mL, 1.00 × 10-4Mol/L Reactive Red 4s operate liquid 1.00mL, and graduation mark is diluted to distilled water, shake up to obtain concentration point
Not Wei 0.05,0.10,0.30,0. 50,1.00,2. 00,3.00,4. 00,5.00,6.00 μ g/mL chitosan it is to be measured molten
Liquid stands 15~120 minutes, synchronizes scanning with λ ex=λ em, records RRS spectrum, and measure ion respectively at 342nm
Associated matterI RRSWith reagent blankI 0, standard curve is drawn, linear equation is further obtained：ΔI = 682.31c+ 114.95
（c, μ g/mL）
In formulaΔI = I RRS-I 0；
Sample detection：The 0.4g samples to be tested acetum of 0.5mol/L is dissolved, 100mL is settled to, is waited for completely molten
It is centrifuged after solution, takes 2.5mL supernatants, and be settled to 100mL, as sample operation liquid, taken 1mL sample operation liquid, be added 2mL's
The B-R buffer solutions that pH is 5.5, add the 1.00 × 10 of 1mL-4Mol/L Reactive Red 4 solution is settled to 10mL with water, mixing
After uniformly, 15~120 minutes are stood, Resonance Rayleigh Scattering intensity is detected, chitosan in sample to be tested is calculated by linear equation
Concentration.Compared with prior art, the present invention has the advantages that：
The present invention has studied Reactive Red 4 and the spectral signature after chitosan association under weakly acidic condition for the first time, and its poly- in shell
Application in sugared quantitative analysis.Result of study shows that in faintly acid B-R buffer systems, the ion of Reactive Red 4 and chitosan is formed
It closes object and generates strong Resonance Rayleigh Scattering characteristic peak, Resonance Rayleigh Scattering intensity and chitosan concentration at λ=342nmcIn good
Good linear relationship, in the concentration range of 0.050~6.00 μ g/mL, linear equation is：ΔI = 682.31c+ 114.95
（c, μ g/mL）,R 2=0.9991,0.033 μ g/mL of detection limit.The present invention utilizes the combination of chitosan and Reactive Red 4 accordingly, establishes
The Resonance Rayleigh Scattering of analysis chitosan, this method have it is easy to operate, quickly, accurately, the features such as high sensitivity.
Description of the drawings
Fig. 1 is ultra-violet absorption spectrum, fluorescence spectrum and Resonance Rayleigh Scattering Spectra.
Fig. 2 is the Resonance Rayleigh Scattering collection of illustrative plates of various concentration chitosan；1. chitosan（2.0μg /mL）；2. Reactive Red 4
（1.0×10-5mol/L）;3~7. Reactive Red 4s (1.0 × 10-5Mol/L)-chitosan (1.0,2.0,3.0,4.0,
5.0 μ g/mL) compound.
Fig. 3 is the influence of active red concentration.
Fig. 4 is the influence of buffer solution dosage.
Fig. 5 is the standard curve and analog sample curve of basic, normal, high molecular weight chitosan；(■) low molecular weight；(●)
Middle-molecular-weihydroxyethyl；(▲) high molecular weight；(▼) middle-molecular-weihydroxyethyl analog sample.
Specific implementation mode
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Chitosan (CTS) standard（At 20 DEG C, 1% is dissolved in 1% acetum）：Low molecular weight is≤200
mPa.S；Middle-molecular-weihydroxyethyl is 200~400mPa.S；High molecular weight is 400~1000mPa.S.Chitosan (CTS) standard solution：Match
It is accurately to weigh 0.0400g chitosans to be dissolved in 0.5mol/L acetums to set method, in volumetric flask constant volume to 100mL, is obtained
The Standard Reserving Solution of a concentration of 400.0 μ g/mL；Storing solution 2.50mL is drawn in the volumetric flask of 100 mL, with water constant volume to quarter
Degree, obtains the chitosan standard solution of 10.00 μ g/mL, now with the current.
Reactive Red 4 operates liquid（1.00×10-4mol/L）：Precision weighs 0.0250g Reactive Red 4s（Sigma companies）Dyestuff,
With water dissolution, the constant volume in 250mL volumetric flasks shakes up, spare.
Britton-Robinson (B-R) buffer solution：By 0.04mol/L mixed acid [(2.71mL orthophosphoric acid+
2.36mL glacial acetic acid+2.47g boric acid)/L] it is formulated by different proportion with 0.2mol/L NaOH solution.
Embodiment 1
One, spectrum analysis：In the colorimetric cylinder of 10mL, 1mL chitosan standard solution is added（10.00μg/mL）, pH be
4.50 B-R buffer solutions 2.00mL, a concentration of 1.00 × 10-4Mol/L Reactive Red 4s operate liquid 1.00mL, and pure water is settled to quarter
Degree, shakes up.Scanning ultravioletvisible absorption light light collection of illustrative plates, excitation spectrum, fluorescence spectrum and Resonance Rayleigh Scattering Spectra respectively, knot
Fruit sees Fig. 1.As shown in Figure 1, CTS- Reactive Red 4s have maximum fluorescence value at excitation wavelength 285nm, 341 nm of wavelength of fluorescence,
The Rayleigh Scattering Spectra of CTS- Reactive Red 4s is located at CTS- Reactive Red 4 absorbing bands, due to Electron absorption electromagnetic wave frequency with
Scattering frequency is identical, and electronics absorbs luminous energy because of resonance and scatters again strongly, generates strong Resonance Rayleigh Scattering, scattering
Intensity improves several orders of magnitude than simple Rayleigh scattering.
There is greater overlap in the shake together maximum scattering position of Rayleigh scattering of fluorescence emission spectral position, this also means that in phase
Under same frequency, resonance and energy exchange can be generated mutually between chitosan and Reactive Red 4, Resonance Rayleigh Scattering is caused to increase
By force, corresponding quenching occurs for fluorescence.
Two, Resonance Rayleigh Scattering collection of illustrative plates
In the colorimetric cylinder of 10mL a series of, sequentially adds the shell that 1mL concentration is respectively 10,20,30,40,50 μ g/mL and gather
Sugar juice, the B-R buffer solutions 2.00mL that pH is 5.50, a concentration of 1.00 × 10-4Mol/L Reactive Red 4s operate liquid 1.00mL, with
Water constant volume shakes up the chitosan solution to be measured for obtaining 1,2,3,4,5 μ g/mL, places 10 min.On F-2500, selective exitation
It is 5nm with transmite slit width, scanning is synchronized with λ ex=λ em, records various concentration chitosan solution（1、2、3、4、5
µg/mL）RRS spectrum, and measure ionic associate respectively at 342nmI RRSWith reagent blankI 0, calculateΔI RRS=I RRS-I 0.As a result Fig. 2 is seen, after as shown in Figure 2, the resonance scattering of chitosan and Reactive Red 4 itself is very weak, but the two combines
Resonance Rayleigh Scattering is remarkably reinforced, this is also due under weakly acidic condition, the amino cation of chitosan and the anion of Reactive Red 4
It is in electroneutral when forming associated matter, hydrophobicity greatly improves, and can cause generating one in conjunction with interface is formed between product and water
The scattering effect of kind surface enhanced, is conducive to RRS enhancings, is in characteristic Resonance Rayleigh Scattering peak at 342nm.This resonance Rayleigh
Scattering strength is linearly related to chitosan concentration.Therefore, subsequent experimental selects the wavelength as Resonance Rayleigh Scattering wavelength.
Three, resonance rayleigh light scattering method experimental condition optimization
1. the selection of buffer solution and pH
B-R buffer solutions, HAc-NaAc buffer solutions, glycine-HCI and six times have been investigated in 1.8~6.5 sections pH
Methyl methylamine-four kinds of hydrochloric acid buffer solution buffer solution is to Resonance Rayleigh Scattering intensity ΔI RRSInfluence, find in B-R and sweet
In two kinds of buffer solution systems of propylhomoserin-hydrochloric acid, the linear relationship of standard series is preferable.But, it is buffered using glycine-HCI molten
Occur the sediment of white when liquid, after standing time 40min in test solution, and compared with glycine-HCI, pH 3.50~
Between 6.50, when using B-R buffer solutions, test solution Resonance Rayleigh Scattering intensity is more stable, when pH value is 5.50ΔIReach most
Big value.Therefore the B-R buffer solutions of subsequent experimental selection pH=5.50.
2. the selection of Reactive Red 4 addition
The colorimetric cylinder of two groups of 10.00mL is taken, one group of chitosan solution for adding a concentration of 50 μ g/mL of 1mL, another group is reagent
Blank, fixed Reactive Red 4 concentration（1.00×10-4mol/L）With B-R pH value of buffer solution, addition（PH=5.50,
2.00mL）, change Reactive Red 4 and operate liquid dosage.It is control with reagent blank, measures the resonance of corresponding Reactive Red 4 addition
Rayleigh intensityI RRS , reagent blankI 0, drawΔI-VCurve, measurement result are shown in Fig. 3.Experiment shows when Reactive Red 4 solution
When dosage is 1.00mL, the Resonance Rayleigh Scattering strength difference of systemΔIIt is maximum.
3. buffer solution addition
The colorimetric cylinder of two groups of 10.00mL is taken, one group of chitosan solution for adding a concentration of 40 μ g/mL of 1mL, another group is reagent
Blank is separately added into 1.00 × 10-4Mol/L Reactive Red 4 solution 1.00mL, B-R buffer solutions fix pH=5.50, change slow
Rush solution usage.The Resonance Rayleigh Scattering intensity of corresponding buffer solution addition is measured, measurement result is shown in Fig. 4.As shown in Figure 4,
When buffer solution addition is 2.00mL, Resonance Rayleigh Scattering intensityΔIIt is maximum.So selecting best buffer solution
2.00mL as experiment condition.
4. order is added
Reagent be added sequence have a certain impact to the combination of substance, so be added order may influence whether to resonate it is auspicious
The intensity of profit scattering.In the colorimetric cylinder of three groups of 10mL, three kinds of addition order are respectively represented：1）Chitosan-B-R buffer solutions-work
Red 4 solution of property；2）Chitosan-Reactive Red 4 solution-B-R buffer solutions；3）B-R buffer solutions-Reactive Red 4-chitosan；Often plus
After entering a kind of reagent and must fully shaking up again plus another reagent, 10min is placed, the RRS that order is added in three groups of differences is measured
Value.Three groups of the variance analysis that order RRS is added is made of SPSS20.0, P ＞ 0.05 illustrate that time ordered pair resonance Rayleigh is added herein
The intensity of scattering does not influence.It tests below and selects to select first scheme as addition order.
5. stabilization time
The colorimetric cylinder of 9 groups of 10.00mL is taken, the chitosan solution of a concentration of 25 μ g/mL of 1mL is successively added, 2mL pH are
5.50 B-R buffer solutions, 1mL a concentration of 1.00 × 10-4Mol/L Reactive Red 4 solution, shakes up, placement 15min, 25min,
35min, 45min, 60min, 90min, 120min, 180min and 240min deduct reagent blank value, measure corresponding standing time
Under Resonance Rayleigh Scattering intensityΔI.It was found that not changing substantially in 120min internal absorbances, have good stability.
Four, linear relationship and detection limit：In the colorimetric cylinder of 10.00mL a series of, it is sequentially separately added into 1mL
Concentration is respectively the chitosan solution of 0.5,1,3,5,10,20,30,40,50,60 μ g/mL, the B-R buffer solutions buffering of pH5.50
Liquid 2.00mL, 1.00 × 10-4Mol/L Reactive Red 4s operate liquid 1.00mL, are diluted to graduation mark with distilled water, shake up, obtain concentration
For the chitosan solution to be measured of 0.050,0.10,0.30,0. 50,1.00,2. 00,3.00,4. 00,5.00,6.00 μ g/mL.
Within the scope of 0.050~6.00 μ g/mL, Resonance Rayleigh Scattering intensity and chitosan concentrationcIn good linear relationship, linear side
Cheng Wei：ΔI = 682.31c+114.95（CTS, μ g/mL）,R 2=0.9991,0.0334 μ g/mL of detection limit.
Five, coexisting substances interfere：Currently, such as crust cellulose capsule of the common health products containing CTS in the market, main auxiliary
Material ingredient has gelatin, starch, dextrin etc..Respectively in the solution of a concentration of 2.00 μ g/mL of chitosan mass, chitin glue is added
Possible coexisting substances in capsule do not carry out interference test measurement, and not plus the standard solution measurement result of interfering substance compare, calculating
The concentration of each coexisting substances is added when generating about ± 5% relative error, the results are shown in Table 1.
1. coexisting substances interference test result of table
| Coexisting substances | Test fluid material concentration（µg/mL） | Relative error (%) |
| Glucose sugar | 5.0×103 | 4.07 |
| Ascorbic acid | 85.00 | 3.18 |
| Citric acid | 15.00 | 4.61 |
| Β-cyclodextrin | 120.00 | 3.58 |
| Soluble starch | 1.00×103 | 4.25 |
| Bovine serum albumin(BSA) | 3.00 | 5.25 |
| Gelatin | 2.00 | -2.34 |
| Glycine | 0.40×103 | -5.04 |
| L-Leu | 60.00 | -1.58 |
| L-Aspartic acid | 1.00 | 2.51 |
| L-lysine | 0.20×103 | -2.53 |
| K+ | 0.45×103 | 2.29 |
| Na+ | 0.60×103 | -5.25 |
| Mg2+ | 0.25 | -3.04 |
| Ca2+ | 2.00 | -1.63 |
| Zn2+ | 1.00×103 | 3.81 |
| Cu2+ | 0.10×103 | -2.43 |
| Fe3+ | 0.20 | 3.33 |
| NH4 + | 883.7 | 4.23 |
The result shows that possible coexisting substances such as soluble starch, citric acid, glucose, glycine in crust cellulose capsule
And the trace element such as potassium, sodium measurement result is interfered it is smaller, on the accuracy of CTS assays in sample influence it is corresponding also compared with
It is small.Part metals ion such as Ca2+、Mg2+、Cu2+And Fe3+It is larger Deng the determination influences to chitosan, it can be by sample test
Suitable EDTA screening agents are added in liquid to eliminate the interference of metal ion.
Seven, the influence of chitosan molecule amount：Take three groups of 10mL colorimetric cylinders, first group of addition low-molecular weight chitoglycan standard,
High molecular weight chitosan standard is added in second group of addition middle-molecular-weihydroxyethyl chitosan standard, third group.The shell of three groups of different molecular weights
The concentration of glycan solution to be measured is 0.05,0.10,0.20,0.30,0.50,1.00,2.00 μ g/mL, while reagent preparation is empty
In vain, each parallel 3 parts.Measure the standard curve of basic, normal, high three kinds of chitosan molecule amounts of various concentration.Use SPSS20.0 pairs
Three standard curves obtain P into line slope significance test<0.05, can tentatively it infer, using Reactive Red 4 as the resonance Rayleigh of probe
Scattering method is influenced by chitosan molecule amount.
On this basis, between the sample curves and above-mentioned three kinds of standard curves of further investigating chitosan analog sample
Otherness.Chitosan analog sample is that 10 μ g/mL of glucose, 5 μ of ascorbic acid are added in the chitosan titer of middle-molecular-weihydroxyethyl
G/mL and 20 μ g/mL chaff interferents of glycine composition.According to the concentration of aforesaid standards curve, the sample for preparing a certain concentration gradient is molten
Liquid, parallel 3 parts, while reagent preparation blank.The fluorescence intensity of each analog sample solution is measured, linear equation is drawn, with three kinds
Molecular weight chitosan standard curve compares, and as a result sees Fig. 5.As seen from Figure 5, analog sample curve and middle-molecular-weihydroxyethyl standard curve
It essentially coincides.
Using SPSS into line slope significance test, the chitosan standard curve and simulation sample of basic, normal, high molecular weight are obtained
It is had differences between 4 curves of product（P<0.05）, and difference is not present between middle-molecular-weihydroxyethyl standard curve and analog sample curve
（P>0.05）.
Analog sample is verified：It is respectively 2.50 μ g/mL and 3.50 μ g/ to take two groups of 10ml colorimetric cylinders, analog sample addition
ML, each parallel three parts, while reagent preparation blank, measure RRS values.Obtain ΔIRespectively with basic, normal, high molecular weight chitosan standard
Chitosan content in the regression equation calculation analog sample of curve, the results are shown in Table 2.
2 same sample of table, three standard curve result of calculations
It is obtained by 2 data of table, when being calculated using middle-molecular-weihydroxyethyl chitosan standard curve, as a result relative error is minimum.According to
This is further verified, and when sample measures, selecting can be maximum as calculating benchmark with the close standard curve of sample curves slope
Degree reduces systematic error.
Application examples 1
Application of the resonance rayleigh light scattering method that the present invention establishes in detection in the market conventional chitosan drug, the application example
The middle conventional medication used is using sharp peacekeeping two brands of Ai get Lan difficult to understand as representative.Ao Liwei has purchased from Weihai Nan Bowan biotechnologys
Limit company, Ai get Lan are purchased from Shanghai Tongji Biological Product Co., Ltd..The detecting step of two kinds of drugs is as follows：
1, sample pretreatment：0. 0400g chitosan capsules powder is accurately weighed respectively（Profit peacekeeping two product of Ai get Lan difficult to understand
Board）, dissolved with the acetum of 0.5mol/L, be settled to 100mL, centrifuged 15min in 6000r/min until completely dissolved, take
The EDTA solution 1mL of 0.01mol/L are added in 2.5mL supernatants, after 100 mL are settled to pure water, as sample operation liquid.
2, sample size measures：First, the sample curves for preparing sharp peacekeeping two kinds of products of Ai get Lan difficult to understand, find two kinds of samples
Curve is suitable with the slope of low-molecular weight chitoglycan standard curve.Therefore, both samples are detected using resonance rayleigh light scattering method
In product when chitosan content, selects low-molecular weight chitoglycan for quantitative criterion, prepare standard curve.
In the colorimetric cylinder of 2 10mL, it is separately added into the sample operation liquid of 1mL sharp peacekeeping two kinds of products of Ai get Lan difficult to understand, often
The B-R buffer solutions 2.00mL, a concentration of 1.00 × 10 that pH is 5.50 are added in a pipe-4The Reactive Red 4 of mol/L operates liquid
1.00mL is settled to graduation mark with water, shakes up and places 10 min.On F-2500, selective exitation and transmite slit width are
5nm synchronizes scanning with λ ex=λ em, records RRS spectrum, and measure ionic associate respectively at 342nmI RRSWith
Reagent blankI 0, calculateΔI RRS=I RRS-I 0。
3, Precision Experiment：According to the method for step 2, the sample operation liquid of certain volume is added in 10 mL colorimetric cylinders,
Parallel 3 parts, while reagent preparation blank and low-molecular weight chitoglycan standard curve.Calculate sample operation liquid chitosan concentration, knot
Fruit is shown in Table 3.
3 sample measurement result of table
| Sample | Ao Liwei（mg/g） | Ai get Lan（mg/g） |
| 1 | 902.5 | 997.5 |
| 2 | 865.0 | 997.5 |
| 3 | 935.0 | 957.5 |
| Average value（mg/g） | 900.8 | 984.2 |
| RSD(%) | 3.88 | 2.35 |
The resonance rayleigh light scattering method rate of recovery is tested：It is separately added into two groups of 10mL colorimetric cylinders with reference to the method for step 2
Then Ao Liwei chitosan sample operation liquid 0.50mL and Ai get Lan chitosan sample operation liquid 1.50mL are respectively managed to Ao Liwei and are added
Enter 10.00 μ g/mL CTS titers 0.30,0.50,1.00mL, the CTS titers of 10.00 μ g/mL are added to each pipes of Ai get Lan
0.50,1.00,2.00mL, each parallel 3 parts, while blank is prepared, each pipe resonance Rayleigh intensity is measured, mark-on reclaims are calculated
Rate.The results are shown in Table 4, obtain the method be averaged recovery of standard addition be 100.0%, precision 3.29%.
4. resonance rayleigh light scattering method recovery of standard addition of table
The resonance rayleigh light scattering method that the present invention makees probe with Reactive Red 4 measures chitosan and the survey of existing resonance rayleigh light scattering method
Determine chitosan research to compare, sensitivity has a distinct increment.Reason may is that Reactive Red 4 molecule has big conjugated structure,
With the rigid plane being connected by azo, the volume for increasing hydrophobic molecule group is more advantageous to compared with linearity molecular structure, to
RRS responses are made to be obviously improved.Influence about chitosan molecule amount to sample measurement result accuracy, resonance Rayleigh dissipate
The method of penetrating can reflect the influence of different molecular weight, therefore, when sample measures, can select the shell close with sample curves slope
Glycan standard curve is as standard, or the concentration of reduction test sample is quantitatively calculated, to reach reduction systematic error, accurately determine
The purpose of amount.
Claims (5)
1. a kind of resonance rayleigh light scattering method of quantitative analysis chitosan, which is characterized in that include the following steps：
The drafting of standard curve：N are configured at the chitosan solution of gradient concentration and 1 blank reagent, to n at gradient concentration
Chitosan solution and 1 blank reagent in be separately added into the B-R buffer solutions of 2 times of volumes, it is then molten to chitosan respectively again
The Reactive Red 4 solution of same volume, the shell at concentration gradient after being diluted after mixing are added in liquid and blank solution
Glycan solution to be measured is stood, and detects the Resonance Rayleigh Scattering intensity of chitosan solution to be measured, is specially carried out with λ ex=λ em same
Step scanning records RRS spectrum, and measures ionic associate respectively at 342nmI RRSWith reagent blankI 0, draw standard
Curve further obtains linear equation：ΔI = 682.31c+ 114.95（c, μ g/mL）
In formulaΔI = I RRS-I 0；
Sample detection：Into the supernatant of the sample to be tested after preliminary treatment, the B-R buffer solutions of 2 times of volumes are added, so
Isometric Reactive Red 4 solution is added afterwards, is stood after mixing, Resonance Rayleigh Scattering intensity is detected, by above-mentioned linear side
Journey calculates the concentration of chitosan in sample to be tested.
2. resonance rayleigh light scattering method according to claim 1, which is characterized in that the time of the standing be 120 minutes with
It is interior.
3. resonance rayleigh light scattering method according to claim 2, which is characterized in that the Reactive Red 4 solution it is a concentration of
1.00×10-4The pH of mol/L, B-R buffer solution is 5.5.
4. resonance rayleigh light scattering method according to claim 3, which is characterized in that the step of sample to be tested preliminary treatment such as
Under：The sample to be tested acetum of 0.5mol/L is dissolved, is centrifuged until completely dissolved, supernatant is obtained, after supernatant dilution
It is detected for fluorescent quenching.
5. a kind of resonance rayleigh light scattering method of quantitative analysis chitosan, which is characterized in that include the following steps：
The drafting of standard curve：In the colorimetric cylinder of 10.00mL a series of, being sequentially separately added into 1mL concentration is respectively
0.5, the chitosan solution of 1,3,5,10,20,30,40,50,60 μ g/mL, the B-R buffer solution buffer solution 2.00mL of pH5.50,
1.00×10-4Mol/L Reactive Red 4s operate liquid 1.00mL, graduation mark is diluted to distilled water, shake up to obtain concentration be respectively
0.05, the chitosan solution to be measured of 0.10,0.30,0. 50,1.00,2. 00,3.00,4. 00,5.00,6.00 μ g/mL, it is quiet
It sets 15~120 minutes, scanning is synchronized with λ ex=λ em, records RRS spectrum, and measure ion association respectively at 342nm
ObjectI RRSWith reagent blankI 0, standard curve is drawn, linear equation is further obtained：ΔI = 682.31c+ 114.95（c, μ
g/mL）
In formulaΔI = I RRS-I 0；
Sample detection：The 0.4g samples to be tested acetum of 0.5mol/L is dissolved, is settled to 100mL, until completely dissolved
Centrifugation, takes 2.5mL supernatants, and be settled to 100mL, as sample operation liquid, takes the 1mL sample operation liquid, the pH that 2mL is added to be
5.5 B-R buffer solutions, add the 1.00 × 10 of 1mL-4Mol/L Reactive Red 4 solution, 10mL is settled to water, is uniformly mixed
Afterwards, 15~120 minutes are stood, detects Resonance Rayleigh Scattering intensity, the dense of chitosan in sample to be tested is calculated by linear equation
Degree.
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| CN106053399A (en) * | 2016-05-27 | 2016-10-26 | 广东药科大学 | Method for measuring chitosan content by using water-soluble aniline blue as probe |
| CN106680062B (en) * | 2017-01-06 | 2019-04-16 | 广东药科大学 | Utilize the method for resonance rayleigh light scattering method measurement anionic surfactant concentration |
| CN109115724A (en) * | 2017-12-30 | 2019-01-01 | 广东药科大学 | A method of to lure the red resonance rayleigh light scattering method for probe to measure chitosan oligosaccharide content |
| CN108956488B (en) * | 2018-04-23 | 2020-11-10 | 山东省医疗器械产品质量检验中心 | Method for measuring protein content in chitosan or chitosan salt |
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| CN102527347A (en) * | 2012-01-19 | 2012-07-04 | 上海海洋大学 | Magnetic chitosan/cationic surface active agent modified zeolite adsorbent and preparation method and application thereof |
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| CN105300945A (en) * | 2015-11-13 | 2016-02-03 | 广东药学院 | Fluorescence quenching method for quantitative analysis of chitosan |
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