CN108760705A - The fluorescence analysis method of benzoyl peroxide in a kind of detection flour - Google Patents
The fluorescence analysis method of benzoyl peroxide in a kind of detection flour Download PDFInfo
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- CN108760705A CN108760705A CN201810570298.8A CN201810570298A CN108760705A CN 108760705 A CN108760705 A CN 108760705A CN 201810570298 A CN201810570298 A CN 201810570298A CN 108760705 A CN108760705 A CN 108760705A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
Abstract
The invention discloses a kind of fluorescence analysis methods of benzoyl peroxide in detection flour, o-phenylenediamine can be aoxidized using the benzoyl peroxide in flour and generate the o-phenylenediamine oxide with fluorescence, can get Determination of benzoyl peroxide in wheat using the method for fluorescence spectrum.The detection of this method benzoyl peroxide is limited to 0.1 μM, and the range of linearity is 0.1~100 μM.
Description
Technical field
The present invention relates to a kind of fluorescence analysis methods of benzoyl peroxide in detection flour.
Background technology
If BPO (i.e. benzoyl peroxide) is excessively added in flour, the benzoic acid of generation easily causes in accumulation human body
Poison.In addition, the decomposition of benzoic acid will carry out in liver, therefore, for the patient of liver function weak people and liver dysfunction,
Its bioconversion hypodynamia, detoxification ability reduce, lead to hepatic disease, easily cause a variety of diseases.Moreover, the oxidation of BPO is made
With the VitAVitE contained in flour, vitamin K and vitamin B1, vitamin B2 etc. can be destroyed, make these vitamins
Content in flour reduces.Vitamin plays an important role during adjusting human metabolism, is that human body is indispensable
Few nutriment.Therefore the extensive concern that the BPO in flour causes researcher how is effectively detected.Currently, detection
The method of BPO mainly has：Iodimetric titration, gas chromatography, high performance liquid chromatography, electrochemical methods, chemoluminescence method, light splitting
Photometry, hexavalent chrome bio-removal, capillary electrophoresis, chromatograph-mass spectrometer coupling method, near infrared spectroscopy etc..
Iodimetric titration reaction principle:BPO is acted on potassium iodide, generates iodine, and iodine is made with quantitative sodium thiosulfate standard solution
With being reduced again, the gauge of the sodium thiosulfate standard solution according to consumption calculates the amount of BPO, and reaction equation is:
C14H10O4+2KI→2C14H10O4K+I2
I2+2Na2S2O3→2NaI+Na2S4O6
Have been reported that display, the detection of the method are limited to 0.102g/kg, recovery of standard addition 95.3% belongs to nonaquepous tration, is tested
Object is dissolved through chloroform methanol, has a large amount of methanol in solution, influence starch coloration, therefore judges terminal with yellow disappearance.
The method is suitable only for the measurement of newly manufacture flour, and sensitivity is relatively low, is not measured to micro BPO inspections.But the method only needs on a small quantity
Glass apparatus and reagent, it is easy to operate, be suitable for lack analytical instrument inspection body of base.
Gas chromatography is the standard method that the flour BPO additive amounts recommended in national standard GB/T18415-2001 measure
One of.In BPO additive amounts in using gas chromatography detection flour, need first to select organic solvent appropriate will be in flour
BPO and its reduzate benzoic acid extract, and BPO therein is then reduced to benzoic acid by suitable reducing agent, by
Gas chromatograph for determination goes out benzoic acid total amount, the last additive amount that BPO is calculated according to the benzoic acid amount measured.Have been reported that display,
When measuring flour sample BPO additive amounts using gas chromatography, BPO and benzoic acid in sample are extracted by extractant of ether,
Iron powder restores the BPO of extraction as reducing agent, measures the BPO rate of recovery between 85%-110%, and detection is limited to 12mg/
kg.And using absolute ethyl alcohol as Extraction solvent, potassium iodide is reducing agent, and the addition of BPO in flour is determined by gas chromatography
Amount, for the detection rate of recovery between 84.3%-108%, detection limit reaches 0.5mg/kg.
High performance liquid chromatography is the standard method that the flour BPO additive amounts recommended in national standard GB/T22325-2008 measure
One of.It has been reported that display, flour sample is extracted using methanol, the BPO extracted is reduced to by benzene by potassium iodide
Formic acid, establishes a kind of high performance liquid chromatography separation detection method of flour BPO additive amounts, and this method rate of recovery can reach
90.1%-102.4%.When measuring flour BPO additive amounts using high performance liquid chromatography, the relative standard deviation of testing result
It is 2.4%~6.8%, detection limit reaches 0.04mg/kg, and the rate of recovery is 86.8%~94.2%.
Electrochemical methods have sensitivity higher, and for minimum analysis detection limit up to 10-12mol/L, accuracy is high, selection
Property is good, and detection speed is fast, and sample requirement is few, handles the advantages that simple, is widely used in bioscience, medical diagnosis, environment
The fields such as detection, food safety detection.It has been reported that display, utilizes benzoyl peroxide in oscillopolarographic detection flour, principle
The benzoic acid generated after carrying out reduction for BPO is after concentrated nitric acid and concentrated sulfuric acid effect, in 0.3M sodium chloride and 1.2M sodium hydroxides bottom
In liquid, when being detected under three-electrode system, occur a derivative peak at -0.7V in oscillographic polarography, the concentration of the peak height and BPO at
Linear relationship, linearly dependent coefficient is between 0.9990~0.9996, and recovery of standard addition is between 92.5%~101.9%.It should
Method precision, accuracy are all higher, while having the characteristics that of low cost, suitable quality inspection station at county level and the application of enterprise's quality inspection room.
And select dichloromethane as extractant, dichloromethane-acetic acid is bottom liquid, and tetrabutylammonium perchlorate is supporting electrolyte, glass carbon
Electrode measures benzoyl peroxide in flour as working electrode, Differential Pulse Voltammetry.Restore peak to peak current and benzoyl peroxide
Formyl concentration is 2.5 × 10-6~10 × 10-4It is in good linear relationship within the scope of mol/L, detection is limited to 2.5 × 10-7mol/
L.The method can be directly used for the measurement of micro benzoyl peroxide in wheat flour, and detect quick, sensitive, accurate, sample use
Amount is less and processing routine is simple.
Chemical energy in the material absorbing chemical reaction process that chemiluminescence refers to and a kind of luminescence phenomenon for generating light radiation.
Chemiluminescent radiant light wavelength and intensity change with chemical reaction difference, therefore the radiant light by being generated to chemiluminescence
The amount of measured matter can be calculated by being detected.This method measures relative deviation and can reach 1.32~2.29%.
Studies have found that in phosphoric acid medium, benzoyl peroxide aoxidizes the iodine and the aobvious blue of starch that potassium iodide generates,
There is maximum absorption band at 585nm wavelength, to establish the micro benzoyl peroxide of iodine indigo plant photometry indirect determination of potassium iodide
Method, such spectrophotometry is easy to operate, quick, cheap, high sensitivity, can be applied to mistake in the wheat flour newly produced
The measurement of Benzoyl Oxide, the rate of recovery is 93.3% or more.
Biosensor is a kind of special sensor, it is by antigen, antibody, protein, the enzyme isoreactivity object after solidification
For matter as sensor sensing element, the variable quantity generated after being reacted it with determinand by energy converter appropriate is converted into telecommunications
Number, then electric signal is carried out to realize the qualitative or quantitative analysis to determinand after corresponding analyzing processing.Studies have found that
Gold electrode surfaces are arrived by gold nanorods and horseradish peroxidase modification are film modified, devise benzoyl peroxide in a kind of detection flour
The biosensor of formyl, the sensor are 0.5%-2.1% to the relative standard deviation that flour benzoyl peroxide detects.It is right
Glass-carbon electrode is sensed using a kind of benzoyl peroxide detection sensor is prepared after silicate gel film and myoglobins modification
Device is 0.5%-2.1% to the detection relative standard deviation of flour sample.
Capillary Electrophoresis, also referred to as high performance capillary electrophoresis are using elastic quartz capillary tube as split tunnel, with high straightening
Galvanic electricity field is driving force, and the electrophoresis point of separation is realized according to the mobility between each component in sample and the difference in distribution behavior
It is one of the Disciplinary Frontiers of Modern Analytical Chemistry research from analysis method.Studies have found that beneficiation technologies and hair will be purged online
Cons electrophoresis combines the additive amount for measuring benzoyl peroxide in flour, the benzoyl peroxide range of linearity that this method measures
For 0.002g/L~0.012g/L, lowest detection is limited to 2mg/L.
Fluorescence method high sensitivity, method easy to operate, and being concerned.
Liquid Chromatography-Mass Spectrometry is a kind of effective detection method that separation analysis can be carried out to complex mixture,
This method has given full play to the component identification capacity for efficiently separating ability and Mass Spectrometer Method of liquid-phase chromatographic analysis, to substance at
The when of being detected is divided to have many advantages, such as that fast detection speed, high sensitivity and sample usage amount are few.Utilizing high performance liquid chromatography-
When Mass Spectrometry measures flour sample, the detection rate of recovery of benzoyl peroxide additive amount is 96.0%-99.3%,
Detection limit is very low, reaches 0.7mg/kg.
Gas chromatography-mass spectrometry is that gas-chromatography piece-rate system and Mass Spectrometer Method system are linked up carry out substance
The method of composition detection, this method can carry out qualitative and quantitative detection to benzoyl peroxide in flour.Powder sample over there
Pre-treating method carry out after accordingly improving, having detected in flour benzoyl peroxide using gas chromatography-mass spectrometry adds
Amount, the minimum detectability of this method reach 0.5 μ g/mL, rate of recovery 95.8%-102.5%.
Near infrared spectroscopy is a kind of respective possessed by near infrared spectrum region based on different measured matter ingredients
Specific spectral quality is come the method that carries out the qualitative and quantitative analysis of material composition.It has been reported that display, utilizes near infrared spectrum
Technology is detected analysis to the additive amount of benzoyl peroxide in flour, using the correlation theory of spectrum analysis to " equivalent is examined
The feasibility of survey " is illustrated, and is verified by experimental result.
Although the addition quantity measuring method of benzoyl peroxide is varied in existing flour, it is difficult to find that one kind simultaneously
With the method that efficient, easy, accuracy is high etc. a little.
Invention content
It is an object of the invention in place of overcome the deficiencies in the prior art, provide benzoyl peroxide first in a kind of detection flour
The fluorescence analysis method of acyl solves the problems in above-mentioned background technology.
The technical solution adopted by the present invention to solve the technical problems is：Provide benzoyl peroxide first in a kind of detection flour
The fluorescence analysis method of acyl, includes the following steps：
(1) metric works curve：O-phenylenediamine solution is added in BR buffer solutions, benzoyl peroxide is added
Solution, 45~55min of water bath with thermostatic control, obtains reaction solution at 35~70 DEG C, by reaction solution and fluorescent spectrophotometer assay its
Standard working curve；0~10 μM of the benzoperoxide solution concentration range, 2 μM of concentration gradient；
(2) sample preparation：Flour is mixed to 0.5~1.5h of ultrasound with ultra-pure water, takes supernatant with 12000~18000r/
The rotating speed of min centrifuges 15~25min, obtains sample solution；
(3) it detects：O-phenylenediamine solution is added in BR buffer solutions, sample solution is added, it is permanent at 35~70 DEG C
45~55min of tepidarium, obtains prepare liquid, is measured by Fluorescence Spectrometer, and by standard working curve, sample solution is calculated
The content of middle benzoyl peroxide.The detection of benzoyl peroxide is limited to 0.1 μM, and the range of linearity is 0.1~100 μM.
In a preferred embodiment of the present invention, the pH of the BR buffer solutions is 6.5~7.5.
In a preferred embodiment of the present invention, the λ of the sepectrophotofluorometerex=415nm, slit 5nm/5nm.
In a preferred embodiment of the present invention, water bath with thermostatic control temperature is 45~55 DEG C in the step (1) and step (3).
In a preferred embodiment of the present invention, the o-phenylenediamine solution a concentration of 1 × 10-5~20 × 10-5M。
In a preferred embodiment of the present invention, the standard working curve is y=9.84x+8.58, R2=0.999, wherein
X is benzoyl peroxide concentration, and y is fluorescence intensity.
In a preferred embodiment of the present invention, the metric works curve includes the following steps：By 50 μ L 5mM's
O-phenylenediamine solution is added in the BR buffer solutions that pH is 7.24, then 0,2 μM, 4 μM, 6 μM, 8 μ are added into colorimetric cylinder respectively
M, 10 μM of 100 μM of benzoyl peroxide solution, control total volume are 1mL；After mixing, reaction solution is anti-in 50 DEG C of waters bath with thermostatic control
50min is answered, its fluorescence spectrum is scanned with Fluorescence Spectrometer immediately after being cooled to room temperature, obtains standard working curve.
Chemical reaction process such as following formula involved by this method：
Compared with the background art, o-phenylenediamine is aoxidized using the benzoyl peroxide in flour and is generated the technical program by it
O-phenylenediamine oxide with fluorescence, using fluorescence spectrum fluorescence intensity to measure Determination of benzoyl peroxide in wheat.This
Method makes full use of the oxidisability of BPO, oxyluciferin reagent o-phenylenediamine (OPD) to generate o-phenylenediamine oxidation product
(OPDox), it generates fluorescence and reaches easy, sensitive and specific detection BPO mesh using Fluorescence Spectrometer fluorescence intensity
, and this method is 0.1 μM -100 μM for the linear response range that BPO is detected.
Description of the drawings
Fig. 1 is to aoxidize the standard working curve that OPD measures BPO with BPO.
Fig. 2 is to aoxidize the selectivity that OPD measures BPO with BPO.
Fig. 3 is the pH condition optimizing curves that BPO aoxidizes that OPD gives birth to glimmering reaction.
Fig. 4 is the temperature condition Optimal Curve that BPO aoxidizes that OPD gives birth to glimmering reaction.
Fig. 5 is the time conditions Optimal Curve that BPO aoxidizes that OPD gives birth to glimmering reaction.
Fig. 6 is the OPD concentration conditions Optimal Curves that BPO aoxidizes that OPD gives birth to glimmering reaction.
Specific implementation mode
Present disclosure is illustrated with reference to the accompanying drawings and examples：
Embodiment 1
One, BPO oxidations OPD is first carried out before detecting and gives birth to glimmering reaction, with optimizing detection condition：
BPO has oxidisability, can be reacted with oxyluciferin reagent o-phenylenediamine (OPD), generates o-phenylenediamine oxidation product
(OPDox), fluorescence is generated
50 μ L 5mM OPD solution are added to 700 μ L 50mM BR (Britton-Robinson) buffer solutions, and (pH is
7.24) in, Britton-Robison buffer solutions are mixed by phosphoric acid, boric acid and acetic acid, and different amounts of hydrogen-oxygen is added thereto
Change sodium, the very wide buffer solution of composition pH ranges.It shakes up, mixed liquor heats 1h at 65 DEG C.It is cooled to room temperature, is used immediately
HITACHI F-7000 Fluorescence Spectrometer measures its fluorescence spectrum (λex=415nm, slit：5nm/5nm).Keep other conditions not
Become, only change system pH (1.98~11.58), research pH gives birth to BPO oxidations OPD the influence of glimmering reaction.Similarly, reaction temperature is investigated
Spend the influence of (30~70 DEG C), reaction time (15~60min), OPD concentration (0~0.5mM) to the catalystic converter system.It is all
Experiment is three groups all parallel.Fig. 3~6 are please referred to, optimal pH, temperature, time and OPD concentration conditions are obtained.
Two, a kind of fluorescence analysis method detecting benzoyl peroxide in flour, includes the following steps：
(1) metric works curve：The OPD solution of 50 μ L 5mM is added in BR buffer solutions (pH 7.24),
100 μM of BPO solution (0,2 μM, 4 μM, 6 μM, 8 μM, 10 μM) of different volumes are added into colorimetric cylinder respectively again, control is overall
Product is 1mL.After mixing, mixed liquor reacts 50min in 50 DEG C of waters bath with thermostatic control, is swept immediately with Fluorescence Spectrometer after being cooled to room temperature
Retouch its fluorescence spectrum (λ ex=415nm, slit：5nm/5nm).All experiments are three groups all parallel, obtain standard working curve, such as
Fig. 1 is measured by detection limit experiment, which is limited to 0.1 μM for the detection of BPO, and the range of linearity is 0.1~100 μM.
(2) sample preparation：
1. buying back two kinds of flour from supermarket at random, 1g flour samples are weighed respectively in the colorimetric cylinder of different 10mL, are used
Ultra-pure water constant volume is to 10mL, ultrasonic 1h；
2. taking supernatant, with rotating speed 15000r/min, 20min is centrifuged, it is spare；
(3) it detects：The OPD solution of 50 μ L 5mM is added in the BR buffer solutions that pH is 7.24, is added into colorimetric cylinder
Sample solution, control total volume are 1mL.After mixing, water bath with thermostatic control 50min, obtains prepare liquid at 50 DEG C, is cooled to room temperature
Afterwards, it is measured by Fluorescence Spectrometer, by standard working curve, the content of benzoyl peroxide in sample solution is calculated.BPO
Detection be limited to 0.1 μM, the range of linearity is 0.1~100 μM.
Three, sample mark-on is tested
(1) sample preparation：
1. buying back two kinds of flour from supermarket at random, 1g flour samples are weighed respectively in the colorimetric cylinder of different 10mL, are used
Ultra-pure water constant volume is to 10mL, ultrasonic 1h；
2. taking supernatant, with rotating speed 15000r/min, 20min is centrifuged, it is spare；
(2) recovery testu operates：
50 μ L 5mM OPD solution are added to the extensive buffer solutions of a certain amount of Britton-Robinson, and (pH is
7.24) in, the prepared 100 μM of BPO solution of 0 μ L, 40 μ L, 60 μ L is added, adds 20 μ L sample solution, control is overall
Product is 1mL.After mixing, mixed liquor reacts 50min in 50 DEG C of waters bath with thermostatic control；Use Fluorescence Spectrometer after being cooled to room temperature afterwards immediately
Survey its fluorescence spectrum (λex=415nm, slit：5nm/5nm).All experiments are three groups all parallel.
As a result such as following table：
1 flour sample of table, 1 mark-on experimental result
2 flour sample of table, 2 mark-on experimental result
Four, BPO aoxidizes the selectivity experiment that OPD measures BPO
Please refer to Fig. 2, the present embodiment also measures the selectivity of BPO with BPO oxidations OPD, as can be seen from Figure, metal from
Son, vitamin B1, vitamin B5 and vitamin C do not respond to the system, and the special inspection of BPO can be realized by demonstrating this method
It surveys.Wherein, the concentration of all metal ions and vitamin is 200 μM.
The above, only present pre-ferred embodiments, therefore cannot limit the scope of implementation of the present invention according to this, i.e., according to
Equivalent changes and modifications made by the scope of the claims of the present invention and description all should still belong in the range of the present invention covers.
Claims (7)
1. the fluorescence analysis method of benzoyl peroxide in a kind of detection flour, which is characterized in that include the following steps：
(1) metric works curve：O-phenylenediamine solution is added in BR buffer solutions, the peroxidating of various concentration is added
Benzoyl solution, 45~55min of water bath with thermostatic control, obtains reaction solution at 35~70 DEG C, measures reaction solution by Fluorescence Spectrometer, obtains
Obtain the standard working curve of benzoyl peroxide；
(2) sample preparation：Flour is mixed to 0.5~1.5h of ultrasound with ultra-pure water, takes supernatant, and with 12000~18000r/
The rotating speed of min centrifuges 15~25min, obtains sample solution；
(3) it detects：O-phenylenediamine solution is added in BR buffer solutions, sample solution is added, the thermostatted water at 35~70 DEG C
45~55min is bathed, prepare liquid is obtained, is measured by Fluorescence Spectrometer, and then by standard working curve, sample solution is calculated
The detection of the content of middle benzoyl peroxide, benzoyl peroxide is limited to 0.1 μM, and the range of linearity is 0.1~100 μM.
2. the fluorescence analysis method of benzoyl peroxide in a kind of detection flour according to claim 1, it is characterised in that：
The pH of the BR buffer solutions is 6.5~7.5.
3. the fluorescence analysis method of benzoyl peroxide in a kind of detection flour according to claim 1, it is characterised in that：
The λ of the sepectrophotofluorometerex=415nm, slit 5nm/5nm.
4. the fluorescence analysis method of benzoyl peroxide in a kind of detection flour according to claim 1, it is characterised in that：
Water bath with thermostatic control temperature is 45~55 DEG C in the step (1) and step (3).
5. the fluorescence analysis method of benzoyl peroxide in a kind of detection flour according to claim 1, it is characterised in that：
The o-phenylenediamine solution a concentration of 1 × 10-5~20 × 10-5M。
6. the fluorescence analysis method of benzoyl peroxide in a kind of detection flour according to claim 1, it is characterised in that：
The standard working curve is y=9.84x+8.58, R2=0.999, wherein x are benzoyl peroxide concentration, and y is that fluorescence is strong
Degree.
7. the fluorescence analysis method of benzoyl peroxide in a kind of detection flour according to claim 1, which is characterized in that
The metric works curve includes the following steps：The o-phenylenediamine solution of 50 μ L 5mM is added to the BR that pH is 7.24 to delay
It rushes in solution, then 0,2 μM, 4 μM, 6 μM, 8 μM, 10 μM of 100 μM of benzoyl peroxide solution, control is added into colorimetric cylinder respectively
Total volume processed is 1mL；After mixing, reaction solution reacts 50min in 50 DEG C of waters bath with thermostatic control, immediately with fluorescence light after being cooled to room temperature
Spectrometer scans its fluorescence spectrum, obtains standard working curve.
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