CN104142378A - Method for semi-quantitative determination of ethyl carbamate through thin layer chromatography - Google Patents
Method for semi-quantitative determination of ethyl carbamate through thin layer chromatography Download PDFInfo
- Publication number
- CN104142378A CN104142378A CN201410365881.7A CN201410365881A CN104142378A CN 104142378 A CN104142378 A CN 104142378A CN 201410365881 A CN201410365881 A CN 201410365881A CN 104142378 A CN104142378 A CN 104142378A
- Authority
- CN
- China
- Prior art keywords
- thin
- urethanes
- fluorescence
- thin layer
- agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a method for semi-quantitative determination of ethyl carbamate through a thin layer chromatography. According to the method, mixed liquor of cinnamyl aldehyde and phosphoric acid is used as a fluorescence derivating agent, mixed liquor of methyl tertiary butyl ether, petroleum ether and methyl alcohol is used as mobile phase developing solvent, a chromatographic sheet is used as an immobile phase, after the ethyl carbamate developed by a thin layer is reacted with the fluorescence derivating agent, accordingly, green fluorescent spots can be observed at the position of 365 nm through a common ultraviolet analysis meter, the Rf value is 0.85, and the lowest drug loading capacity of a thin layer plate is 50 ng of ethyl carbamate. According to the limiting standard of 5 mg/L ethyl carbamate in wine, 2.5 mg/L EC can be detected directly, and the sample application amount of the thin layer plate is generally 20 microliters maximally. The method is small in number of needed samples, rapid, high in sensitivity, simple in step, small in number of needed equipment and easy to operate. In accordance with the principle, a rapid test kit can be researched and developed, and the method is used for rapidly detecting the ethyl carbamate in alcoholic beverages and fermented food.
Description
Technical field
The present invention relates to a kind of method that detects urethane ester content, be specifically related to the method for thin-layered chromatography semiquantitative determination urethane ester content.
Background technology
Urethanes (Ethyl Carbamate is called for short EC) is a kind of compound with potential carcinogenesis, can cause the diseases such as liver cancer, cutaneum carcinoma, lung cancer and lymph cancer, is the product of following of fermented food and alcoholic beverage.EC is just proved to be carcinogen as far back as nineteen forty-three.1976, Ough(Ough C S. Ethyl carbamate in fermented beverages and foods. II. Possible formation of ethyl carbamate from diethyl dicarbonate additionto wine [J]. Agric FoodChem, 1976,24 (2): 328-331.) find to contain EC in alcoholic beverage.Research shows, to rodent, EC is a kind of multidigit point carcinogenic substance, and ethanol (alcohol) has facilitation to its carcinogenicity.2007, international cancer research institute changed EC as 2A group (may make us the material that class is suffered from cancer) to the mankind's carcinogenic toxicity into by 2B group (maybe may make us the material that class is suffered from cancer).The pollution of urethanes at present, is considered to food relaying aflatoxin another major issue afterwards, and China not yet formulates the limit standard of urethanes in alcoholic beverage and fermented food.
Find EC in fermented alcohol drink since, be all devoted to easy, EC detection method of content research fast and accurately both at home and abroad, the pre-treating method of sample and detection method are all being updated.At present the instrumental analysis detection method of EC in alcoholic beverage is had to vapor-phase chromatography, gas chromatography-mass spectrography, liquid phase chromatography, liquid chromatography-mass spectrography, thin-layered chromatography, Fourier transform infrared spectrometry etc. both at home and abroad, wherein the most frequently used is gas chromatography-mass spectrography.Above-mentioned detection method accuracy is high, but there is instrument and equipment costliness, pre-treating method complexity, the shortcoming such as personnel specialty technical requirement is high, testing cost is high.Therefore, set up easy, fast, detection method becomes EC in alcoholic beverage and detects important content in the urgent need to address accurately.
Simple to operate, quick, the highly sensitive and feature such as accurately and reliably that thin-layer chromatography has, is widely used in food nutrition, synthetic drug and drug metabolism, medical science and clinical, toxicological analysis and judicial chemistry, the residual detection of agriculture etc.For using thin-layered chromatography to detect urethanes, report actually rare both at home and abroad.The domestic power of the taking in the fresh (power of taking in the fresh, Chen Jiehua, Dai Haixiang. the tlc analysis of urethanes [J] in wine. health research, 1991, 20 (6): 45-46.) use thin-layered chromatography directly to launch under 365nm, the detection of method is limited to 10mg/L, external Eugenie Jaryj(Eugenie Jaryj, Klemens Lorenz, Spangenberg B. A Simple Method for the Quantification of Urethane in Spirits[J]. Journal of Liquid Chromatography & Related Technologies. 2008, 31:1969-1976.) use thin-layered chromatography, after launching, react with EC derivating agent, result visible blue phosphor dot.
The invention provides a kind of method for fast detecting urethanes.The research of this method based on Eugenie Jaryj, after improving, can use more cheap thin layer plate, after launching, react with cinnamic acid derivating agent, result can be at general ultraviolet analyser 365nm, Rf value is that 0.85 place observes green fluorescence point, the urethanes that the minimum medicine carrying amount of thin layer plate is 50ng.If according to the limit standard of urethanes 5mg/L in wine, the point sample amount that the present invention can direct-detection goes out the EC(thin layer plate of 2.5mg/L is generally 20 μ L to the maximum), therefore the present invention can be fast, simple, cheapness detects at least urethanes of 2.5mg/L.
Summary of the invention
The invention provides the method for thin-layered chromatography semiquantitative determination urethanes.Do fluorescence derivating agent with cinnamic acid, phosphoric acid mixed liquor, taking methyl tert-butyl ether, sherwood oil, methyl alcohol mixed liquor as mobile phase developping agent, chromatographic sheet is fixing phase, after the urethanes of thin-layer developing reacts with fluorescence derivating agent, at uv analyzer (365nm place, dark situation) can be observed visually green urethanes fluorescence spot, Rf value is fixing;
Concrete steps are:
(1) take out thin-layer silicon offset plate and in 105 DEG C ~ 110 DEG C baking ovens, activate half an hour, put into exsiccator for subsequent use;
(2) preparation developping agent, pours into after stirring in double flute thin layer chromatograph developing cylinder, leaves standstill 0.5 ~ 1 hour, makes it saturated;
(3) preparation fluorescence derivating agent solution: by cinnamic acid, acetone, phosphoric acid mixes, for subsequent use after stirring;
(4) apart from 1cm place, thin-layer silicon offset plate lower end point sample, on same side apart from 2cm place, thin-layer silicon offset plate upper end pencil mark as launching frontal line, point sample sample is the urethanes liquid to be measured that at least contains 50ng;
(5) in thin-layer developing cylinder, launch, developping agent arrives and launches to take out after frontal line, dries;
(6) after the thin layer plate after drying being dipped in fluorescence derivating agent, take out, afterwards again baking oven reaction 5-20 minutes;
(7) under 365nm place dark situation condition, observe thin layer plate at uv analyzer, now thin layer plate background color is blue and white, if the exhibition that has a urethanes at point has green spot clearly according to scope, this spot is the fluorescence spot after urethanes derives, if when the urethane ester content containing in point sample amount is less than 50ng, spot is invisible.
In said method, in described step (1), described thin-layer silicon offset plate is Qingdao Haiyang thin-layer silicon offset plate, and its specification is G or GF, 5cm × 10cm or 10cm × 10cm.
In said method, developping agent described in step (2) consist of methyl tert-butyl ether: sherwood oil (high boiling range) and methyl alcohol, its volume ratio is methyl tert-butyl ether: sherwood oil (high boiling range): methyl alcohol=(3-6): (3-10): (1-5); Preferable methyl tertbutyl ether: sherwood oil (high boiling range): methyl alcohol=(4-6): (5-7): (2-4).
In said method, described in step (3), fluorescent color-developing agent consumption is respectively, cinnamic acid 20 μ L-1mL, acetone 10-80ml, phosphoric acid 0.1-10ml; Preferably cinnamic acid is 60-200 μ L, and acetone is 30-50mL, and phosphoric acid is 2-5ml.
In said method, the point sample amount of the urethane ester content described in step (4) in sample on thin-layer silicon offset plate is at least 50ng.
In said method, the time in fluorescence derivating agent that is dipped in described in step (6) was 1 ~ 20 second, preferably 1 ~ 3 second; Reaction, in 80-150 DEG C, baking oven, preferably 100-140 DEG C, is reacted preferably 5-12 minutes 5-20 minutes.
Thin-layered chromatography of the present invention compared with prior art, has the following advantages:
(1) the present invention does not need can detect at least urethanes of 2.5mg/L with large-scale instrument, and Rf value is 0.85.
(2) sample required for the present invention is few, with low cost, and step is simple, and reaction conditions is easy to control, and equipment needed thereby is few, easy operating.
(3) because urethanes is very easily water-soluble and ethanol, when thin-layer chromatography, spot is very easy to diffusion, so select suitable developping agent to make spot in best observation place and to reduce diffusional effect extremely important, the developping agent that this method is selected has been attempted the detection for mark-on wine urethanes, result shows, clear spot, naked eyes Observable.
Embodiment
Below in conjunction with embodiment, the present invention is described in more detail: the Rf value of addressing in embodiment, it is the fixed constant that in thin-layer developing, analysans launches in specific fixing phase and mobile phase, by comparing with the Rf value of standard items, can carry out qualitative analysis to material to be separated.In the present invention, after expansion, need to react with derivating agent, observe at 365nm place, measure.Calculate Rf value by following formula:
In formula: initial point is the position of initial point sample; Spot is the sample spot after thin-layer developing.
Embodiment 1:
(1) take out thin-layer silicon offset plate 5cm × 10cm and activate half an hour in 110 DEG C of baking ovens, put into exsiccator for subsequent use;
(2) preparation developping agent (methyl tert-butyl ether: sherwood oil: methyl alcohol=5:7:3(volume ratio), pours into after stirring in double flute thin layer chromatograph developing cylinder, leaves standstill at least half an hour, makes it saturated;
(3) preparation fluorescence derivating agent solution, cinnamic acid 160 μ L, acetone 40ml, phosphoric acid 2.4ml is in small beaker, for subsequent use after stirring;
(4) take out thin layer plate, apart from 1cm place, thin layer plate lower end point sample, apart from 2cm place, thin layer plate upper end pencil mark as expansion forward position, point sample.The first sample spot is 20 μ L urethanes standard solution (5mg/L, 40% ethanol water), putting second point of horizontal 2cm place's point apart from first, sample is the urethanes standard solution (10mg/L of 20 μ L, 40% ethanol water) and urea liquid (10mg/L, 40% ethanol water) volume ratio 1:1 mixed liquor; Urethanes be purity more than 99% purchased from Aladdin reagent company.
(5) in thin-layer developing cylinder, launch, developping agent takes out after arriving line forward position, dries;
(6) after the thin layer plate after drying being dipped in rapidly in fluorescence derivating agent, take out, afterwards again 130 DEG C of reactions of baking oven 10 minutes;
(7) at uv analyzer (365nm place, dark situation) observation thin layer plate, high-visible blue and white background, the first top, point sample place has green fluorescence point (Rf value is 0.85), the second point sample place to have green fluorescence point (Rf value is 0.85) and blue-fluorescence point (Rf value is 0.26).; urea can be separated through thin-layer developing with urethanes; due to different component under same chromatographic condition because the difference of the polarity of molecule own can move to the differing heights of thin layer plate; measure respectively the distance of initial point to spot center; initial point is to the distance of solvent front; according to Rf value computing formula, with the Rf value contrast of standard items, can carry out qualitative analysis.
Embodiment 2:
(1) take out thin-layer silicon offset plate 5cm × 10cm and activate half an hour in 110 DEG C of baking ovens, put into exsiccator for subsequent use;
(2) preparation developping agent methyl tert-butyl ether: sherwood oil: methyl alcohol=5:7:3(volume ratio), after stirring, pour in thin-layer developing cylinder, leave standstill at least half an hour, make it saturated;
(3) preparation fluorescence derivating agent solution, cinnamic acid 160 μ L, acetone 40ml, phosphoric acid 2.4ml is in small beaker, for subsequent use after stirring;
(4) take out thin layer plate, apart from 1cm place, thin layer plate lower end point sample, apart from 2cm place, thin layer plate upper end pencil mark as expansion forward position, point sample.The first sample spot is 20 μ L urethanes standard solution (5mg/L, 40% ethanol water), putting second point of horizontal 2cm place's point apart from first, sample is 20 μ L urethanes standard solution (10mg/L, 40% ethanol water) and commercially available dragon side's Three-Star wine (1:1) mixed liquors; Urethanes be purity more than 99% purchased from Aladdin reagent company;
(5) in thin-layer developing cylinder, launch, developping agent takes out after arriving line forward position, dries;
(6) after the thin layer plate after drying being dipped in rapidly in fluorescence derivating agent, take out, afterwards again 130 DEG C of reactions of baking oven 10 minutes;
(7) at uv analyzer (365nm place, dark situation) observation thin layer plate, high-visible blue and white background, the first top, point sample place has green fluorescence point (Rf value is 0.85), the second point sample place to have green fluorescence to select (Rf value is 0.85) and the brandy through thin-layer developing.That is, brandy can be separated through thin-layer developing with urethanes, and the compound in brandy does not affect substantially on EC thin-layer developing.
The above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here without also giving exhaustive to all embodiments.All any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in the protection domain of the claims in the present invention.
Claims (9)
1. the method for thin-layered chromatography semiquantitative determination urethanes, it is characterized in that, do fluorescence derivating agent with cinnamic acid, phosphoric acid mixed liquor, taking methyl tert-butyl ether, sherwood oil, methyl alcohol mixed liquor as mobile phase developping agent, chromatographic sheet is fixing phase, after the urethanes of thin-layer developing reacts with fluorescence derivating agent, can be observed visually green urethanes fluorescence spot at uv analyzer, Rf value is fixing; Concrete steps are:
(1) take out thin-layer silicon offset plate and in 105 DEG C ~ 110 DEG C baking ovens, activate half an hour, put into exsiccator for subsequent use;
(2) preparation developping agent, pours into after stirring in double flute thin layer chromatograph developing cylinder, leaves standstill 0.5 ~ 1 hour, makes it saturated;
(3) preparation fluorescence derivating agent solution: by cinnamic acid, acetone, phosphoric acid mixes, for subsequent use after stirring;
(4) apart from 1cm place, thin-layer silicon offset plate lower end point sample, on same side apart from 2cm place, thin-layer silicon offset plate upper end pencil mark as launching frontal line, point sample sample is the urethanes liquid to be measured that at least contains 50ng;
(5) in thin-layer developing cylinder, launch, developping agent arrives and launches to take out after frontal line, dries;
(6) after the thin layer plate after drying being dipped in fluorescence derivating agent, take out, afterwards again baking oven reaction 5-20 minutes;
(7) under 365nm place dark situation condition, observe thin layer plate at uv analyzer, now thin layer plate background color is blue and white, if the exhibition that has a urethanes at point has green spot clearly according to scope, this spot is the fluorescence spot after urethanes derives, if when the urethane ester content containing in point sample amount is less than 50ng, spot is invisible.
2. the method for thin-layered chromatography semiquantitative determination urethanes according to claim 1, is characterized in that, in described step (1), described thin-layer silicon offset plate is Qingdao Haiyang thin-layer silicon offset plate, and its specification is G or GF, 5cm × 10cm or 10cm × 10cm.
3. the method for thin-layered chromatography semiquantitative determination urethanes according to claim 1, is characterized in that, developping agent described in step (2) consist of methyl tert-butyl ether, sherwood oil (high boiling range) and methyl alcohol; Its volume ratio is methyl tert-butyl ether: sherwood oil (high boiling range): methyl alcohol=(3-6): (3-10): (1-5).
4. the method for thin-layered chromatography semiquantitative determination urethanes according to claim 1, is characterized in that, developping agent described in step (2) consist of methyl tert-butyl ether, sherwood oil (high boiling range) and methyl alcohol; Its volume ratio is methyl tert-butyl ether: sherwood oil (high boiling range): methyl alcohol==(4-6): (5-7): (2-4).
5. the method for thin-layered chromatography semiquantitative determination urethanes according to claim 1, is characterized in that, described in step (3), fluorescent color-developing agent consumption is respectively, cinnamic acid 20 μ L-1mL, acetone 10-80ml, phosphoric acid 0.1-10ml.
6. the method for thin-layered chromatography semiquantitative determination urethanes according to claim 1, is characterized in that, described in step (3), fluorescent color-developing agent consumption is respectively, and cinnamic acid is 60-200 μ L, and acetone is 30-50mL, and phosphoric acid is 2-5ml.
7. the method for thin-layered chromatography semiquantitative determination urethanes according to claim 1, is characterized in that, the point sample amount of the urethane ester content described in step (4) in sample on thin-layer silicon offset plate is at least 50ng.
8. the method for thin-layered chromatography semiquantitative determination urethanes according to claim 1, is characterized in that, described in step (6), being dipped in the time in fluorescence derivating agent was 1 ~ 20 second, reacts for reacting 5-20 minutes in 80-150 DEG C, baking oven.
9. the method for thin-layered chromatography semiquantitative determination urethanes according to claim 1, is characterized in that, described in step (6), being dipped in the time in fluorescence derivating agent was 1 ~ 3 second, reacts for reacting 5-12 minutes in 100-140 DEG C, baking oven.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410365881.7A CN104142378B (en) | 2014-07-29 | 2014-07-29 | The method of thin-layered chromatography semiquantitative determination urethanes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410365881.7A CN104142378B (en) | 2014-07-29 | 2014-07-29 | The method of thin-layered chromatography semiquantitative determination urethanes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104142378A true CN104142378A (en) | 2014-11-12 |
| CN104142378B CN104142378B (en) | 2015-12-30 |
Family
ID=51851612
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410365881.7A Expired - Fee Related CN104142378B (en) | 2014-07-29 | 2014-07-29 | The method of thin-layered chromatography semiquantitative determination urethanes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104142378B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101959865A (en) * | 2008-02-28 | 2011-01-26 | 诺瓦提斯公司 | Quinolines as farnesyl pyrophosphate synthase inhibitor |
| CN103323415A (en) * | 2013-07-01 | 2013-09-25 | 广州市酒类检测中心 | Enzyme inhibition method for detecting carbamates |
-
2014
- 2014-07-29 CN CN201410365881.7A patent/CN104142378B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101959865A (en) * | 2008-02-28 | 2011-01-26 | 诺瓦提斯公司 | Quinolines as farnesyl pyrophosphate synthase inhibitor |
| CN103323415A (en) * | 2013-07-01 | 2013-09-25 | 广州市酒类检测中心 | Enzyme inhibition method for detecting carbamates |
Non-Patent Citations (4)
| Title |
|---|
| EUGENIE JARYJ ET AL: "A Simple Method for the Quantification of Urethane in Spirits", 《JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES》, vol. 31, no. 13, 12 August 2008 (2008-08-12) * |
| 吴世嘉 等: "发酵食品中氨基甲酸乙酯的研究进展", 《化学与生物工程》, vol. 26, no. 9, 25 September 2009 (2009-09-25) * |
| 梁新红 等: "酒精饮料中氨基甲酸乙酯检测方法研究进展", 《食品工业科技》, no. 8, 1 August 2010 (2010-08-01) * |
| 罗杰 等: "氨基甲酸乙酯检测方法研究进展", 《酿酒科技》, no. 8, 18 August 2012 (2012-08-18) * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104142378B (en) | 2015-12-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230142304A1 (en) | Methods for cannabinoid quantification | |
| Kostelanska et al. | The study of deoxynivalenol and its masked metabolites fate during the brewing process realised by UPLC–TOFMS method | |
| Ajtony et al. | Determination of ethyl carbamate in wine by high performance liquid chromatography | |
| Li et al. | Rapid determination of ethanol in fermentation liquor by full evaporation headspace gas chromatography | |
| Merli et al. | From smart drugs to smartphone: A colorimetric spot test for the analysis of the synthetic cannabinoid AB-001 | |
| Kächele et al. | NMR investigation of acrolein stability in hydroalcoholic solution as a foundation for the valid HS-SPME/GC–MS quantification of the unsaturated aldehyde in beverages | |
| Agatonovic-Kustrin et al. | Analysis of phenolics in wine by high performance thin-layer chromatography with gradient elution and high resolution plate imaging | |
| Zeng et al. | Fast visual monitoring of the freshness of beef using a smart fluorescent sensor | |
| CN104655753B (en) | The assay method of 3-acetyl group-2,5-thioxene in a kind of food additive | |
| Zhang et al. | Sensitive determination of melamine leached from tableware by reversed phase high-performance liquid chromatography using 10-methyl-acridone-2-sulfonyl chloride as a pre-column fluorescent labeling reagent | |
| Han et al. | A novel fluorescent probe for formaldehyde based-on monomer-excimer conversion and its imaging in live cells | |
| Lv et al. | Development of an efficient HPLC fluorescence detection method for brassinolide by ultrasonic-assisted dispersive liquid–liquid microextraction coupled with derivatization | |
| Walther et al. | Development of brewing science in (and since) the late 19th century: molecular profiles of 110–130 year old beers | |
| CN101285813A (en) | Taurolidine quality checking method | |
| Luo et al. | Detection of primary aromatic amines content in food packaging ink and migration from printed plastic bags | |
| Liu et al. | A novel method for the determination of the ethanol content in soy sauce by full evaporation headspace gas chromatography | |
| Li et al. | Determination of biogenic amines in apples and wine with 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4, 4-difluoro-1, 3, 5, 7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene by high performance liquid chromatography | |
| Al-Shaalan | Determination of phenylephrine hydrochloride and chlorpheniramine maleate in binary mixture using chemometric-assisted spectrophotometric and high-performance liquid chromatographic-UV methods | |
| CN104142378B (en) | The method of thin-layered chromatography semiquantitative determination urethanes | |
| CN104237230B (en) | The quick determination method of epiphysin and its operation vessel combination | |
| CN103399111B (en) | Method for selectively measuring ethylene glycol monoethyl ether acetate in dry food packaging paper based on headspace-gas chromatography/mass spectrometry | |
| CN102830114A (en) | Detection method for content of tannin in plant extract liquid | |
| CN114965751A (en) | Method for measuring chemical component content in myrobalan | |
| CN104502486A (en) | Method for determining methyl vanillin and ethyl vanillin in milk powder by adopting headspace-solid phase microextraction technology | |
| Horii et al. | Determination of ethyl carbamate in Sake using headspace solid phase microextraction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151230 Termination date: 20210729 |