阅读说明：本技术 油类物质中邻苯二甲酸酯类增塑剂的荧光法检测方法 (The Fluorometric assay method of phthalic ester plasticizer in oily substance ) 是由 殷龙龙 李群 许光映 高忠东 王振 嵇莎莎 张倩茹 尹蓉 胡晓军 于 2019-09-27 设计创作，主要内容包括：本发明公开了一种油类物质中邻苯二甲酸酯类增塑剂的荧光法检测方法,包括(1)制备邻苯二甲酸酯标准曲线；(2)制备皂化水解液；(3)制备荧光衍生物；(4)检测与换算。本发明采用NaOH将油类物质完全皂化、将邻苯二甲酸酯类水解成邻苯二甲酸钠,再通过加入石油醚在离心的作用下将油脂和溶解有邻苯二甲酸钠的水溶液彻底分开,然后在过氧化氢的作用下将邻苯二甲酸钠中和为邻苯二甲酸,并将具有给电子效应的过氧根(-O-)结构结合到邻苯二甲酸的羧基上,从而使本来存在于油脂里的不能产生荧光的邻苯二甲酸酯被分离出来并且产生荧光,使得采用荧光检测手段成为可能。本操作简便、使用试剂较少、检测条件温和易控,成本低廉,工作曲线线性范围宽。(The invention discloses a kind of Fluorometric assay methods of phthalic ester plasticizer in oily substance, including (1) to prepare phthalic acid ester standard curve；(2) saponification liquid is prepared；(3) fluorescent derivative is prepared；(4) detection and conversion.The present invention is fully saponified by oily substance using NaOH, phthalate is hydrolyzed into sodium phthalate, grease and aqueous solution dissolved with sodium phthalate are thoroughly separated under the effect of centrifugation by the way that petroleum ether is added again, then under the action of hydrogen peroxide by sodium phthalate and be phthalic acid, and peroxide root (- O-) structure with electron effect is integrated on the carboxyl of phthalic acid, to which the phthalic acid ester that cannot generate fluorescence made to be originally present in grease is separated and generates fluorescence, make it possible using fluorescence detection means.This is easy to operate, less using reagent, testing conditions are mildly easily-controllable, and low in cost, the working curve range of linearity is wide.)

1. the Fluorometric assay method of phthalic ester plasticizer, includes the following steps: in a kind of oily substance

(1) it prepares phthalic acid ester standard curve and weighs phthalic acid ester standard items 1mg, 2mg, 3mg, 4mg, 5mg respectively In 5 1L volumetric flasks by number, with triglycerides constant volume, it is configured to a series of O-phthalics of content distribution gradient Acid esters standard solution, then from pipetting 1mL in the standard solution of 5 1L volumetric flasks respectively into the 20mL container of number consecutively, to It is separately added into the NaOH solution of 10mL10% in all containers, then all containers are placed in water-bath, constant temperature stirs at 90-100 DEG C It is evaporated to liquid, is saponified sample to be tested sufficiently, the phthalic acid ester contained by it is fully hydrolyzed into sodium phthalate and alcohol, It then cools to room temperature, then is separately added into 10mL deionized water into container, stir, make a small amount of O-phthalic generated in container Sour sodium is substantially soluble in water, is stood 1h, is then taken 3mL lower clear liquid to be placed in centrifuge tube respectively from all containers, and to centrifugation 2mL petroleum ether is separately added into pipe, sealing centrifuge tube, which is centrifuged to solution, to be obviously layered, and is divided from centrifuge tube after centrifugation Clear liquid 1mL is not removed, is respectively placed in the colorimetric cylinder of identical number, and the hydrogen peroxide of 1mL30% is separately added into colorimetric cylinder Solution shakes up, and all colorimetric cylinders are placed in insulating box together, keeps the temperature 1-2h under the conditions of 35-90 DEG C, is subsequently cooled to room Temperature carries out the measurement of emitted luminescence intensity with sepectrophotofluorometer to the solution in all colorimetric cylinders, finally by phthalic acid The corresponding emitted luminescence intensity value of ester standard solution and the content of the phthalic acid ester in standard solution carry out linear fit, obtain The standard curve and equation of phthalic acid ester content and emitted luminescence intensity corresponding relationship；

(2) the oily substance sample to be measured that saponification liquid measure takes 1mL is prepared, moves in 20mL container, is added into container 10% NaOH solution 10mL, container is placed in water-bath, and constant temperature, which is stirred to liquid, at 90-100 DEG C is evaporated, after being cooled to room temperature 10mL deionized water is added into container, stirring is substantially soluble in a small amount of sodium phthalate generated in container in water, stands 1h；

(3) prepare fluorescent derivative takes 3mL lower clear liquid to be placed in centrifuge tube from the solution that (2) are handled, and adds into centrifuge tube Enter 2mL petroleum ether, sealing centrifuge tube, which is centrifuged to solution, to be obviously layered, and removes clear liquid 1mL after centrifugation from centrifuge tube It is placed in the colorimetric cylinder of (1) same size, the hydrogenperoxide steam generator of 1mL30% is added, shakes up and is placed in insulating box, in 35- Keep the temperature 1-2h under the conditions of 90 DEG C, after be cooled to room temperature；

(4) it detects and the fluorescent derivatization solution of (3) preparation is carried out emitted luminescence intensity measurement with sepectrophotofluorometer with conversion, It will test the content that result substitutes into phthalic acid ester in the standard curve and equation calculating sample to be tested that (1) obtains.

Technical field

The invention belongs to detection technique fields, and in particular to one kind phthalate suitable for oily substance is plasticized The detection method of agent.

Background technique

Phthalic acid ester (Phthalate esters, PAEs), usually by phthalic acid and 4~15 carbon One group of ester type compound that alcohol is formed, at normal temperature, in addition to minority is the solid of low melting point, generally higher boiling low volatility Thick liquid, it is colorless and transparent, there is special odor, be insoluble in water, be soluble in a variety of organic solvents such as ether, methanol and ethyl alcohol, belongs to Liposoluble substance.Because its excellent performance in terms of enhancing plasticity is widely used as plasticiser applied to packaging for foodstuff material In the production of the various plastic products such as material, medical instruments, cosmetics.However PAEs is not connected with covalent bond and plastic matrix molecule It connects, the two is only to be connected with hydrogen bond or Van der Waals force.PAEs is in free state in the plastic, when contacting the substances such as water, grease It is easy to happen migration, so that External Environment, such as water, soil and atmosphere pollute, organism can also be impacted.PAEs Belong to interferon in biology, although toxicity is less, enrichment power is strong, and long-term intake will affect normal point of biological hormone in vivo It secretes, brings harm to immune system, reproductive system and digestive system, it is also possible to induced hepatocellular carcinoma and the number or knot for causing chromosome Structure mutation.Because PAEs belongs to liposoluble substance, the phthalic acid ester when installing oils product with plastic products, in plastics Substance is just easy to be slowly released into oil, is not finally eaten person's intake in vivo, gradually damages to body.Now, Europe Alliance, the U.S., Japan, the Chinese blacklist that phthalate substance is all successively included in priority pollutants, the U.S. has 6 kinds of DEHP, BBP, DBP, DOP, DEP, DMP, China has 3 kinds of DEP, DMP, DOP.

Foreign countries for PAEs research starting it is more early, it is domestic then relatively late.Laboratory and industry are used for the inspection of PAEs at present Survey method mainly has gel permeation chromatography, gas chromatography, gas chromatography mass spectrometry method, gas chromatography tandem mass spectrometry method, efficient liquid phase Chromatography, Liquid Chromatography-Tandem Mass Spectrometry etc..However these instrumental methods are operationally all comparatively laborious, detection cycle is longer, Professional degree is high, and needs to put into biggish cost, is suitable for large and medium-sized enterprise or special testing agency, is unfavorable for small miniature enterprise The development of industry, and if can largely reduce the fixed cost of enterprise using fluorescence rule.CN105866089 B is public Having opened " a kind of method of Fluorometric assay phthalic ester plasticizer ", it is first used in tetrahydrofuran dissolving test sample Phthalic acid ester；Dioxane and strong base solution is added, phthalic acid ester is hydrolyzed into phthalate, purification hydrolysis Afterwards, upper layer aqueous solution is taken, hydrochloric acid solution, PBS solution, copper nitrate solution and hydrogenperoxide steam generator, phthalic acid are sequentially added Salt is converted to the hydroxyl phthalic salt that can be fluoresced, and manganese dioxide and strong base solution are added after reaction, stands or is centrifuged, protects It stays supernatant aqueous solution as solution to be measured, finally solution to be measured is measured with fluorescence method.The invention can not need large size Under conditions of detection device, the phthalic acid ester content for plastics, toy, painting layer, packaging material for food etc. is detected. But the invention is not suitable for phthalic acid ester content in grease and detects, and the chemical reagent for detecting needs is on the high side, operation step Rapid also more complicated.

Summary of the invention

In view of the problems of the existing technology, the present invention provides one kind phthalate suitable for oily substance and increases Mould the Fluorometric assay method of agent, it is intended to realize in Fluorometric assay oily substance by using relatively easy easy operation Phthalic ester plasticizer.

The present invention is achieved through the following technical solutions.

The Fluorometric assay method of phthalic ester plasticizer, includes the following steps: in a kind of oily substance

(1) it prepares phthalic acid ester standard curve and weighs phthalic acid ester standard items 1mg, 2mg, 3mg, 4mg, 5mg respectively In 5 1L volumetric flasks by number, with triglycerides constant volume, it is configured to a series of O-phthalics of content distribution gradient Acid esters standard solution.Again from pipetting 1mL in the standard solution of 5 1L volumetric flasks respectively into the 20mL container of number consecutively, to It is separately added into the NaOH solution of 10mL10% in all containers, then all containers are placed in water-bath, constant temperature stirs at 90-100 DEG C It is evaporated to liquid, is saponified sample to be tested sufficiently, the phthalic acid ester contained by it is fully hydrolyzed into sodium phthalate and alcohol, It then cools to room temperature, then is separately added into 10mL deionized water into container, stir, make a small amount of O-phthalic generated in container Sour sodium is substantially soluble in water, is stood 1h, is then taken 3mL lower clear liquid to be placed in centrifuge tube respectively from all containers, and to centrifugation 2mL petroleum ether is separately added into pipe, sealing centrifuge tube, which is centrifuged to solution, to be obviously layered, and is divided from centrifuge tube after centrifugation Clear liquid 1mL is not removed, is respectively placed in the colorimetric cylinder of identical number, and the hydrogen peroxide of 1mL30% is separately added into colorimetric cylinder Solution shakes up, and all colorimetric cylinders are placed in insulating box together, keeps the temperature 1-2h under the conditions of 35-90 DEG C, is subsequently cooled to room Temperature carries out the measurement of emitted luminescence intensity with sepectrophotofluorometer to the solution in all colorimetric cylinders, finally by phthalic acid The corresponding emitted luminescence intensity value of ester standard solution and the content of the phthalic acid ester in standard solution carry out linear fit, obtain The standard curve and equation of phthalic acid ester content and emitted luminescence intensity corresponding relationship.

(2) the oily substance sample to be measured that saponification liquid measure takes 1mL is prepared, is moved in 20mL container, is added into container The NaOH solution 10mL for entering 10%, container is placed in water-bath, and constant temperature, which is stirred to liquid, at 90-100 DEG C is evaporated, and is cooled to room temperature 10mL deionized water is added in backward container, stirring is substantially soluble in a small amount of sodium phthalate generated in container in water, quiet Set 1h.

(3) prepare fluorescent derivative takes 3mL lower clear liquid to be placed in centrifuge tube from the solution that (2) are handled, and to centrifuge tube Middle addition 2mL petroleum ether, sealing centrifuge tube, which is centrifuged to solution, to be obviously layered, and removes clear liquid after centrifugation from centrifuge tube 1mL is placed in the colorimetric cylinder of (1) same size, and the hydrogenperoxide steam generator of 1mL30% is added, shakes up and is placed in insulating box, In 1-2h is kept the temperature under the conditions of 35-90 DEG C, after be cooled to room temperature.

(4) it detects and the fluorescent derivatization solution of (3) preparation is carried out emitted luminescence intensity survey with sepectrophotofluorometer with conversion It is fixed, it will test the content that result substitutes into phthalic acid ester in the standard curve and equation calculating sample to be tested that (1) obtains.

The content detection of phthalic acid ester of this method suitable for oily substance.

Fluorescence detection is a kind of advanced analysis method, because its device therefor is compared with simple, sensitivity for analysis is high, selectivity is strong With use simplicity, have compared to mass spectrography, spectroscopic methodology, polarography etc. and widely apply.Fluorescence detection can directly and The numerous organic compounds of Analysis of Grounding and inorganic elements are usually applied in many necks such as food industry and biological medicine industry Domain.Conjugated system or rigid plane with stronger cloud density are the necessary conditions that substance generates fluorescence.However, in oil There are two the carboxyls with sucting electronic effect on the molecular structure of phthalate in substance, to reduce phenyl ring On cloud density, cause it that fluorescence detection is directly used to occur without the peak of obvious transmitting light.

The beneficial effects of the present invention are: (1) it is using NaOH that oily substance is fully saponified, by phthalate water Solution is at sodium phthalate, then passes through and petroleum ether is added under the effect of centrifugation by grease and dissolved with the water of sodium phthalate Solution thoroughly separates, to realize the plasticiser detection in oils product.(2) then under the action of hydrogen peroxide by adjacent benzene two It is phthalic acid that sodium formate, which neutralizes, and peroxide root (- O-) structure with electron effect is integrated to phthalic acid On carboxyl, so that the phthalic acid ester that cannot generate fluorescence for making to be originally present in grease is separated and generates glimmering Light makes it possible using fluorescence detection means.(3) this is easy to operate and less using reagent, the working curve range of linearity Width, in addition to sepectrophotofluorometer, the present invention does not need that the adjacent benzene two of detection can be completed under conditions of large-scale detection device investment The work of formate ester plasticizer, easy to operate and low in cost, testing conditions are mildly easily-controllable.

Detailed description of the invention

Fig. 1 is the phthalic acid ester content of embodiment 1 and the standard curve of emitted luminescence intensity corresponding relationship.

Fig. 2 is the phthalic acid ester content of embodiment 2 and the standard curve of emitted luminescence intensity corresponding relationship.

Embodiment 1

Using the method for the content of phthalic acid ester in the method for the present invention detection sesame oil.

(1) prepare phthalic acid ester standard curve weigh respectively phthalic acid ester standard items 1mg, 2mg, 3mg, 4mg, 5mg, with triglycerides constant volume, are configured to a series of of content distribution gradient in 5 1L volumetric flasks by number Phthalic acid ester standard solution.The 20mL for pipetting 1mL to number consecutively respectively from the standard solution of 5 1L volumetric flasks again holds In device, the NaOH solution of 10mL10% is separately added into Xiang Suoyou container, then all containers are placed in water-bath, constant temperature at 90 DEG C Stirring is evaporated to liquid, is saponified sample to be tested sufficiently, the phthalic acid ester contained by it is fully hydrolyzed into sodium phthalate And alcohol, it then cools to room temperature, then be separately added into 10mL deionized water into container, stirs, make a small amount of neighbour generated in container Phthalic acid sodium is substantially soluble in water, is stood 1h, is then taken 3mL lower clear liquid to be placed in centrifuge tube respectively from all containers, and 2mL petroleum ether is separately added into centrifuge tube, sealing centrifuge tube, which is centrifuged to solution, to be obviously layered, from centrifugation after centrifugation Clear liquid 1mL is removed in pipe respectively, is respectively placed in the colorimetric cylinder of identical number, and is separately added into the mistake of 1mL30% into colorimetric cylinder Hydrogen peroxide solution shakes up, and all colorimetric cylinders are placed in insulating box together, keeps the temperature 2h under the conditions of 35 DEG C, is subsequently cooled to room Temperature carries out the measurement of emitted luminescence intensity with sepectrophotofluorometer to the solution in all colorimetric cylinders, the transmitting that will finally obtain Light intensity value and the content of the phthalic acid ester in corresponding standard solution carry out linear fit, obtain phthalic acid ester and contain The standard curve (such as Fig. 1) and equation of amount and emitted luminescence intensity corresponding relationship:

Y=20.52X-8.068, R²=0.994。

Y is represented emitted luminescence intensity (unit: A.U.), and X represents the concentration (unit: mg/L) of phthalic acid ester.

(2) it prepares saponification liquid measure and takes 1mL brand sesame oil, move in 20mL container, 10% is added into container NaOH solution 10mL, container is placed in water-bath, and constant temperature, which is stirred to liquid, at 90 DEG C is evaporated, and is added after being cooled to room temperature into container Enter 10mL deionized water, stir, be substantially soluble in a small amount of sodium phthalate generated in container in water, stands 1h.

(3) prepare fluorescent derivative takes 3mL lower clear liquid to be placed in centrifuge tube from the solution that (2) are handled, and to centrifuge tube Middle addition 2mL petroleum ether, sealing centrifuge tube, which is centrifuged to solution, to be obviously layered, and removes clear liquid after centrifugation from centrifuge tube 1mL is placed in the colorimetric cylinder of (1) same size, and the hydrogenperoxide steam generator of 1mL30% is added, shakes up and is placed in insulating box, In It is cooled to room temperature after keeping the temperature 2h under the conditions of 35 DEG C.

(4) it detects and the fluorescent derivatization solution of (3) preparation is carried out emitted luminescence intensity survey with sepectrophotofluorometer with conversion It is fixed, result 15.64A.U..It will test the Y that result substitutes into equation, calculate adjacent benzene in sample to be tested i.e. certain brand edible oil The content X of dicarboxylic acid esters is about 1.1554mg/L.