KR100537403B1 - Determination Method of Amitrole in Water Samples - Google Patents
Determination Method of Amitrole in Water Samples Download PDFInfo
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- KR100537403B1 KR100537403B1 KR10-2003-0040045A KR20030040045A KR100537403B1 KR 100537403 B1 KR100537403 B1 KR 100537403B1 KR 20030040045 A KR20030040045 A KR 20030040045A KR 100537403 B1 KR100537403 B1 KR 100537403B1
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- South Korea
- Prior art keywords
- amitrol
- gas chromatography
- isobutyl chloroformate
- water
- present
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- KLSJWNVTNUYHDU-UHFFFAOYSA-N Amitrole Chemical compound NC1=NC=NN1 KLSJWNVTNUYHDU-UHFFFAOYSA-N 0.000 title claims abstract description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 10
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims abstract description 19
- 238000001212 derivatisation Methods 0.000 claims abstract description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims description 9
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 claims 4
- 239000012468 concentrated sample Substances 0.000 claims 2
- 230000008020 evaporation Effects 0.000 claims 1
- 238000004817 gas chromatography Methods 0.000 abstract description 19
- 238000004458 analytical method Methods 0.000 abstract description 14
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 239000004009 herbicide Substances 0.000 abstract description 3
- 230000002363 herbicidal effect Effects 0.000 abstract description 2
- 238000004949 mass spectrometry Methods 0.000 abstract description 2
- 238000004904 shortening Methods 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
- 239000003960 organic solvent Substances 0.000 description 13
- 238000001514 detection method Methods 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000000605 extraction Methods 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 239000003651 drinking water Substances 0.000 description 4
- 235000020188 drinking water Nutrition 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- QAEDZJGFFMLHHQ-UHFFFAOYSA-N trifluoroacetic anhydride Chemical compound FC(F)(F)C(=O)OC(=O)C(F)(F)F QAEDZJGFFMLHHQ-UHFFFAOYSA-N 0.000 description 4
- BBEAQIROQSPTKN-LHNTUAQVSA-N 1,2,3,4,5,6,7,8,9,10-decadeuteriopyrene Chemical compound [2H]C1=C([2H])C([2H])=C2C([2H])=C([2H])C3=C([2H])C([2H])=C([2H])C4=C([2H])C([2H])=C1C2=C43 BBEAQIROQSPTKN-LHNTUAQVSA-N 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- RENMDAKOXSCIGH-UHFFFAOYSA-N Chloroacetonitrile Chemical compound ClCC#N RENMDAKOXSCIGH-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- RIFGWPKJUGCATF-UHFFFAOYSA-N ethyl chloroformate Chemical compound CCOC(Cl)=O RIFGWPKJUGCATF-UHFFFAOYSA-N 0.000 description 2
- 239000001307 helium Substances 0.000 description 2
- 229910052734 helium Inorganic materials 0.000 description 2
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 2
- HVTICUPFWKNHNG-UHFFFAOYSA-N iodoethane Chemical compound CCI HVTICUPFWKNHNG-UHFFFAOYSA-N 0.000 description 2
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- PVWOIHVRPOBWPI-UHFFFAOYSA-N n-propyl iodide Chemical compound CCCI PVWOIHVRPOBWPI-UHFFFAOYSA-N 0.000 description 2
- DGTNSSLYPYDJGL-UHFFFAOYSA-N phenyl isocyanate Chemical compound O=C=NC1=CC=CC=C1 DGTNSSLYPYDJGL-UHFFFAOYSA-N 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000002098 selective ion monitoring Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- XMTQQYYKAHVGBJ-UHFFFAOYSA-N 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA Chemical compound CN(C)C(=O)NC1=CC=C(Cl)C(Cl)=C1 XMTQQYYKAHVGBJ-UHFFFAOYSA-N 0.000 description 1
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 1
- 239000005510 Diuron Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- FZFAMSAMCHXGEF-UHFFFAOYSA-N chloro formate Chemical compound ClOC=O FZFAMSAMCHXGEF-UHFFFAOYSA-N 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 229910052805 deuterium Inorganic materials 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- ZFKJVJIDPQDDFY-UHFFFAOYSA-N fluorescamine Chemical compound C12=CC=CC=C2C(=O)OC1(C1=O)OC=C1C1=CC=CC=C1 ZFKJVJIDPQDDFY-UHFFFAOYSA-N 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003673 groundwater Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 238000000752 ionisation method Methods 0.000 description 1
- 125000005929 isobutyloxycarbonyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])OC(*)=O 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000006207 propylation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 238000004454 trace mineral analysis Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7206—Mass spectrometers interfaced to gas chromatograph
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1826—Organic contamination in water
- G01N33/184—Herbicides, pesticides, fungicides, insecticides or the like
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/306—Pesticides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4022—Concentrating samples by thermal techniques; Phase changes
- G01N2001/4027—Concentrating samples by thermal techniques; Phase changes evaporation leaving a concentrated sample
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/025—Gas chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/12—Preparation by evaporation
- G01N2030/126—Preparation by evaporation evaporating sample
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
본 발명은 수질 시료 내에 잔류하는 제초제인 아미트롤을 분석하는 방법에 관한 것으로, 보다 구체적으로는 수질 시료를 직접 진공회전증발기로 완전히 증발 및 농축시킨 후, 이소부틸 클로로포르메이트를 사용해서 실온에서 15분 내지 20분간 반응시켜 유도체화하고, 기체 크로마토그래피(GC) 또는 기체 크로마토그래피(GC)/질량 분석기(MS)를 사용하여 아미트롤의 농도를 분석하는 단계를 포함하는 이미트롤의 검출법에 관한 것이다. 본 발명의 방법에 따라 수질 시료에서 아미트롤의 농도를 분석할 경우 우수한 감도, 분석 시간의 단축, 간편성 및 경제성을 동시에 얻을 수 있다.The present invention relates to a method for analyzing amitrol, a herbicide remaining in a water sample. More specifically, the water sample is completely evaporated and concentrated by a direct vacuum rotary evaporator, and then isobutyl chloroformate is used at room temperature. And derivatization by reacting for minutes to 20 minutes, and analyzing the concentration of amitrol using gas chromatography (GC) or gas chromatography (GC) / mass spectrometry (MS). . According to the method of the present invention, when analyzing the concentration of amitrol in a water sample, excellent sensitivity, shortening of analysis time, simplicity and economy can be simultaneously obtained.
Description
본 발명은 음용수와 같은 수질 시료에서 대표적 제초제인 아미트롤(amitrole, 3-아미노-1,2,4-트리아졸)을 검출하는 검출법에 관한 것으로, 보다 상세하게는 아미트롤을 진공회전증발기를 이용하여 농축한 후, 일정한 반응 시간 및 온도 하에서 이소부틸 클로로포르메이트 (isobutyl chloroformate, iso-BCF)와 반응시켜 유도체화한 후, 이를 직접 기체 크로마토그래피(GC) 또는 기체 크로마토그래피/질량 분석기(GC/MS)를 사용하여 분석함으로써 아미트롤을 분석하는 방법에 관한 것이다. 본 발명의 방법은 우수한 감도, 분석 시간의 단축, 간편성, 경제성 등의 장점이 있어, 수질에서 미량으로 잔류하는 농약의 모니터링에 효과적으로 널리 응용될 수 있다.The present invention relates to a detection method for detecting amitrole (amitrole, 3-amino-1,2,4-triazole), which is a representative herbicide in a water sample such as drinking water. Concentrated, and then derivatized with isobutyl chloroformate (isoo-BCF) under constant reaction time and temperature, and then directly derivatized with gas chromatography (GC) or gas chromatography / mass spectrometry (GC / MS) to analyze amitrol. The method of the present invention has advantages such as excellent sensitivity, shortening of analysis time, simplicity, and economic efficiency, and thus can be effectively applied to monitoring pesticides remaining in trace amounts in water quality.
본 발명에서 분석하고자 하는 아미트롤은 디우론과 같이 금지된 제초제의 대용으로 널리 사용되고 있다. 그러나, 아미트롤은 물에 대한 용해도가 매우 크기 때문에 환경 내 유입될 경우 지하수와 지표수를 오염시키고, 결과적으로 음용수의 오염을 초래한다 (I. Bobeldijk, K. Broess, P. Speksnijder, T. van Leerdam, J. Chromatogr. A, 938(2001) 15-22).Amitrol to be analyzed in the present invention is widely used as a substitute for prohibited herbicides such as diuron. However, Amitrol has a very high solubility in water, which, when introduced into the environment, contaminates groundwater and surface water, resulting in contamination of drinking water (I. Bobeldijk, K. Broess, P. Speksnijder, T. van Leerdam). , J. Chromatogr. A, 938 (2001) 15-22).
또한, 이 물질은 휘발성이 낮고 물에는 매우 잘 녹는 반면에 일반적인 유기 용매에는 거의 녹지 않아 (메틸렌 클로라이드에는 약 11mg/ℓ, 헥산의 경우에는 1mg/ℓ) 추출이 매우 어렵다. 또한, 분자 내에 여러 개의 아미노기(-NH)를 가지고 있어서 극성이 큰 편으로, 아미트롤을 그대로 분석하는 경우에는 미량 분석에 문제가 있거나, 기체 크로마토그래피로는 검출이 불가능한 경우가 있다.In addition, the material is low in volatility and very soluble in water, while insoluble in common organic solvents (about 11 mg / l in methylene chloride and 1 mg / l in hexane), making extraction very difficult. In addition, since there are several amino groups (-NH) in a molecule | numerator, since it is large in polarity, when analyzing an amitrol as it is, there exists a problem in trace analysis, or it may be impossible to detect by gas chromatography.
이러한 이유로 인하여 아미트롤의 분석을 위해선 주로 플루오레스카민 (fluorescamine)을 이용하여 아미트롤을 유도체화한 후 액체 크로마토그래피를 이용하여 분석하는 방법이 사용되어 왔다. 하지만, 플루오레스카민을 이용한 유도체화에 의한 분석 방법은 플루오레스카민을 과량을 사용해야 하고 실험 절차가 복잡하고 까다로운 등 여러 가지 문제점이 있다.For this reason, for the analysis of amitrol, a method of derivatizing amitrol mainly using fluorescamine and then using liquid chromatography has been used. However, the analytical method by derivatization using fluorescarmine has various problems such as excessive use of fluorescarmine and complicated and difficult experimental procedures.
따라서, 신속하고 경제적이며 미량까지 분석이 가능한 안정적인 아미트롤의 검출법이 여전히 요구되고 있다.Therefore, there is still a need for a stable method of detecting amitrol that can be analyzed quickly, economically and in trace amounts.
본 발명자들은 상기 문제점들을 해결하고자 예의 연구한 결과, 음용수와 같은 수질 시료를 직접 증발 및 농축시킨 후 이소부틸 클로로포르메이트를 사용하여 유도체화한 후, 이를 직접 기체 크로마토그래피 또는 기체 크로마토그래피/질량 분석기를 사용하여 아미트롤을 분석할 경우 신속하고 경제적으로 아미트롤을 미량의 농도까지 분석할 수 있다는 것을 발견하여 본 발명에 이르게 되었다.The present inventors have diligently studied to solve the above problems, and as a result, the water samples, such as drinking water, are directly evaporated and concentrated, and then derivatized using isobutyl chloroformate, which is then directly subjected to gas chromatography or gas chromatography / mass spectrometry. In the analysis of amitrol using the present invention was found to be able to quickly and economically analyze amitrol to a trace concentration.
따라서, 본 발명의 목적은 음용수와 같은 수질 시료를 직접 증발 및 농축시킨 후 이소부틸 클로로포르메이트를 사용하여 유도체화한 후, 이를 직접 기체 크로마토그래피 또는 기체 크로마토그래피/질량 분석기를 사용하여 아미트롤을 분석하는 방법을 제공하는 것이다.Accordingly, it is an object of the present invention to directly evaporate and concentrate a water sample, such as drinking water, to derivatize it with isobutyl chloroformate, which is then directly purified using a gas chromatography or gas chromatography / mass spectrometer. It provides a way to analyze.
이하, 본 발명에 따른 아미트롤의 검출법에 대해 상세하게 설명하겠다.Hereinafter, the detection method of amitrol according to the present invention will be described in detail.
본 발명에 따른 아미트롤의 검출법은 수질 시료의 농축 단계, 이소부틸 클로로포르메이트에 의한 유도체화 단계, 및 기체 크로마토그래피 또는 기체 크로마토그래피/질량 분석기에 의한 아미트롤의 분석 단계를 포함한다.The detection method of amitrol according to the present invention includes the step of concentrating the water sample, derivatization with isobutyl chloroformate, and analyzing the amitrol by gas chromatography or gas chromatography / mass spectrometry.
본 발명에서 분석하고자 하는 아미트롤은 유기 용매에 대한 추출율이 매우 낮기 때문에 유기 용매로 추출하는 데에는 어려움이 있다. 이에 따라, 본 발명에서는 유기 용매를 사용하여 추출하지 않고 5 내지 10㎖의 수질 시료를 직접 진공회전증발기로 완전히 증발 및 농축시킨다.Amitrol to be analyzed in the present invention is difficult to extract with an organic solvent because the extraction rate for the organic solvent is very low. Accordingly, in the present invention, 5-10 ml of the water sample is completely evaporated and concentrated by a direct vacuum rotary evaporator without extraction using an organic solvent.
이와 같이 농축된 수질 시료를 하기 반응식 1에 나타낸 바와 같이 이소부틸 클로로포르메이트와 반응시켜 유도체화한다.The water sample thus concentrated is derivatized by reaction with isobutyl chloroformate as shown in Scheme 1 below.
상기 유도체화 반응에서 사용되는 이소부틸 클로로포르메이트의 양은 40 ~ 60㎕가 적당하며, 유도체화 반응 시간 및 반응 온도는 각각 15분 내지 20분 및 실온이 적당하나, 이에 제한되는 것은 아니다.The amount of isobutyl chloroformate used in the derivatization reaction is appropriately 40 to 60 μl, and the derivatization reaction time and reaction temperature are suitably 15 minutes to 20 minutes and room temperature, respectively, but are not limited thereto.
또한, 상기 유도체화 반응에서, 농축된 수질 시료를 150 ~ 250㎕의 메틸렌 클로라이드에 용해시킨 후 이소부틸 클로로포르메이트를 첨가하여 유도체화 반응을 수행할 수 있다.In addition, in the derivatization reaction, the concentrated water sample may be dissolved in 150 to 250 μl of methylene chloride, and then isobutyl chloroformate may be added to perform the derivatization reaction.
이와 같이 유도체화한 후 수득된 결과물을 기체 크로마토그래피 또는 기체 크로마토그래피/질량 분석기를 사용하여 아미트롤을 분석한다.The result obtained after this derivatization is analyzed for amitrol using gas chromatography or gas chromatography / mass spectrometry.
상기 기체 크로마토그래피에서 사용되는 이동상은 기체 크로마토그래피에서 일반적으로 사용되는 모든 기체가 가능하며, 바람직하게는 산화에 불활성인 기체, 즉 질소, 헬륨 및 수소 등이 이동상으로 사용될 수 있다. 또한, 본 발명에 따라 사용될 수 있는 컬럼의 경우는 컬럼의 길이와 내경에는 특별한 제한이 없으며 단지 중간 정도 극성이면 된다.The mobile phase used in the gas chromatography may be any gas generally used in gas chromatography, and preferably, gases which are inert to oxidation, ie, nitrogen, helium and hydrogen, may be used as the mobile phase. In addition, in the case of a column that can be used according to the present invention, there is no particular limitation on the length and the inner diameter of the column, but only a moderate polarity.
이와 같은 본 발명에 따른 아미트롤의 검출법은 플루오레스카민을 이용한 아미트롤의 검출법에 비하여 절차가 단순하고 신속하며 경제적이다. 또한, 플루오레스카민을 이용한 아미트롤의 검출법의 경우 일반적으로 검출 한계가 수질 시료 20㎖에 대하여 약 1㎕/ℓ(일본 SPEED '98의 액체 크로마토그래피/형광 검출기를 이용한 방법)인 반면, 본 발명에 따른 아미트롤의 검출법의 검출 한계는 수질 시료 10㎖에 대하여 0.1㎕/ℓ로서, 본 발명에 따른 아미트롤의 검출법은 향상된 감도 및 선택성을 제공한다.The detection method of amitrol according to the present invention is simpler, faster and more economical than the detection method of amitrol using fluorescarmine. In addition, in the case of the detection of amitrol using fluorescarmine, the detection limit is generally about 1 μl / L for the 20 ml of the water sample (method using a liquid chromatography / fluorescence detector of SPEED '98, Japan). The detection limit of the amitrol detection method according to the present invention is 0.1 µl / L for 10 ml of the water sample, and the detection method of the amitrol according to the present invention provides improved sensitivity and selectivity.
이하, 하기 실시예로 본 발명을 보다 구체적으로 설명하겠지만, 본 발명은 어떠한 식으로든 하기 실시예에 제한되지 않는다.Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples in any way.
<실시예 1><Example 1>
8개의 10㎖ 시험관에 아미트롤의 표준물질 용액 (메탄올 1㎖ 당 100㎍) 및 내부표준물질인 중수소로 치환된 피렌-d10 용액 (아세트산에틸 1㎖ 당 100㎍)을 각각 100㎕ 및 10㎕씩 첨가하였다.100 μl and 10 μl of Amitrol standard solution (100 μg per 1 mL of methanol) and pyrene-d 10 solution (100 μg per 1 mL of ethyl acetate) substituted with internal deuterium in 8 10 mL test tubes Added freshly.
8개의 시험관 중 3개의 시험관에 아세톤 150㎕ 및 탄산칼륨 분말 약 2-3mg을 첨가한 후, 요오드화메틸, 요오드화에틸 또는 요오드화프로필을 각각 50㎕씩 첨가하였다. 또 다른 하나의 시험관에는 트리플루오로무수초산 100㎕와 트리플루오로에탄올 50㎕를 첨가하였다. 상기 4개의 시험관을 80℃에서 1시간 동안 반응시켰다.150 µl of acetone and about 2-3 mg of potassium carbonate powder were added to three of the eight tubes, followed by 50 µl of methyl iodide, ethyl iodide or propyl iodide, respectively. In another test tube, 100 μl of trifluoroacetic anhydride and 50 μl of trifluoroethanol were added. The four test tubes were reacted at 80 ° C. for 1 hour.
요오드화메틸, 요오드화에틸 또는 트리플루오로무수초산과 트리플루오로에탄올을 첨가한 시험관은 반응이 일어나지 않았으며, 요오드화프로필을 첨가한 시험관은 프로필화 반응이 일어났지만, 아미트롤에 존재하는 1차 아미노기 뿐만 아니라 2차 아미노기에도 프로필화 반응이 일어나 분석 결과 여러 개의 피크 (peak)가 검출되어 아미트롤의 정성 및 정량 분석에는 적합하지 않았다.Test tubes with methyl iodide, ethyl iodide or trifluoroacetic anhydride and trifluoroethanol did not react. Test tubes with propyl iodide did not react, but only the primary amino groups present in the amitrol. In addition, the secondary amino group was also subjected to a propylation reaction, and as a result, several peaks were detected, which was not suitable for qualitative and quantitative analysis of amitrol.
또한, 나머지 4개의 시험관에 각각 모노클로로아세토니트릴, 페닐 이소시아네이트, 에틸클로로포르메이트 또는 이소부틸 클로로포르메이트를 각각 50㎕씩 첨가한 후 실온에서 20분간 반응시켰다.In addition, 50 µl of monochloroacetonitrile, phenyl isocyanate, ethylchloroformate or isobutyl chloroformate was added to the remaining four test tubes, respectively, and reacted at room temperature for 20 minutes.
상기 4개의 시험관 중 모노클로로아세토니트릴, 페닐 이소시아네이트 또는 에틸클로로포르메이트를 첨가한 3개의 시험관에서는 반응이 일어나지 않았으나, 이소부틸 클로로포르메이트를 첨가한 시험관은 반응이 일어났다. 이를 분석한 결과, 이소부틸 클로로프르메이트를 첨가한 경우에는 이소부톡시 카르보닐기 (-COOCH2CH(CH3)2)가 아미트롤의 1개의 1차 아미노기에서만 반응이 일어나 하나의 피크만 검출된 것을 확인할 수 있었다.The reaction did not occur in the three test tubes to which monochloroacetonitrile, phenyl isocyanate or ethylchloroformate was added, but the test tube to which isobutyl chloroformate was added occurred. As a result of the analysis, when isobutyl chloroformate was added, the isobutoxy carbonyl group (-COOCH 2 CH (CH 3 ) 2 ) reacted only with one primary amino group of amitrol, and only one peak was detected. Could.
<실시예 2><Example 2>
본 실시예는 추출 용매에 따른 아미트롤의 추출율을 보기 위한 것이다.This example is for viewing the extraction rate of amitrol according to the extraction solvent.
4개의 25㎖의 분액 깔대기에 10㎖의 증류수를 넣고, 아미트롤의 표준물질 용액 (메탄올 1㎖ 당 100㎍) 및 내부표준물질인 피렌-d10 용액 (아세트산에틸 1㎖ 당 100㎍)을 각각 100㎕ 및 10㎕씩 첨가하였다.10 ml of distilled water was added to four 25 ml separatory funnels, and a standard solution of amitrol (100 µg per 1 ml of methanol) and a pyrene-d 10 solution (100 µg per 1 ml of ethyl acetate) were used as internal standards. 100 μl and 10 μl each were added.
4개의 분액 깔대기 중 3개의 분액 깔대기에는 염석 효과를 꾀하기 위하여 2g 정도의 무수 황산나트륨을 넣어준 후, 각 분액 깔대기에 디에틸에테르 (ether), 메틸렌 클로라이드 (MC) 또는 메틸터셔리부틸에테르 (MTBE)를 10㎖씩을 각각 첨가하고, 15분간 진탕한 후 추출하였다. 추출 후의 물 층을 제거한 다음 무수 황산나트륨 약 0.5g을 첨가하여 유기 용매 층의 물기를 완전히 제거하였다. 이와 같이 물기를 완전히 제거한 각 유기 용매 층을 질소 건조기를 이용하여 모두 증발시켜 농축하였다.Three of the four funnels were added with about 2 g of anhydrous sodium sulfate for salting effect, and then the diluent ether, methylene chloride (MC) or methyl tertiary butyl ether (MTBE) was added to each funnel. 10 ml each was added, shaken for 15 minutes and extracted. After removing the water layer after extraction, about 0.5 g of anhydrous sodium sulfate was added to completely dry the organic solvent layer. Thus, each organic solvent layer which completely removed water was concentrated by evaporating using the nitrogen drier.
각 농축물을 메틸렌 클로라이드 200㎕에 녹인 후 이소부틸 클로로포르메이트 50㎕를 각각 첨가하여 실온에서 20분 동안 유도체화 반응을 실시하였다.Each concentrate was dissolved in 200 µl of methylene chloride, and 50 µl of isobutyl chloroformate was added thereto, followed by derivatization at room temperature for 20 minutes.
추출 과정을 거치지 않은 나머지 1개 시료는 직접 진공회전증발기를 사용하여 수분을 완전히 제거한 후 메틸렌 클로라이드 200㎕에 용해시킨 다음 이소부틸 클로로포르메이트 50㎕를 첨가하여 실온에서 20분 동안 유도체화 반응을 실시하였다.The remaining one sample was not completely extracted using direct vacuum rotary evaporator, dissolved in 200 μl of methylene chloride, and then 50 μl of isobutyl chloroformate was added to conduct derivatization at room temperature for 20 minutes. It was.
유도체화 반응 후 수득한 상기 4개의 시료를 질소 하에서 건조시킨 후, 각 시료를 아세트산에틸 용액 100㎕에 다시 용해시킨 후 직접 기체 크로마토그래피에 2㎕씩 주입하여 분석하였다.The four samples obtained after the derivatization reaction were dried under nitrogen, and each sample was dissolved in 100 µl of ethyl acetate solution and injected directly into 2 µl of gas chromatography for analysis.
각 분석 결과를 도 1에 나타내었다. 도 1에서 볼 수 있는 바와 같이, 유기 용매로 추출하지 않고 진공회전증발기를 이용하여 직접 농축한 경우 아미트롤의 높은 회수율을 나타내었으나, 유기 용매를 사용하여 추출하고 농축할 경우에는 그 회수율이 상당히 낮아 유기 용매에 아미트롤이 거의 추출이 되지 않았음을 알 수 있었다.Each analysis result is shown in FIG. As can be seen in Figure 1, the direct recovery using a vacuum rotary evaporator without extraction with an organic solvent showed a high recovery of amitrol, but when the extraction and concentration using an organic solvent is significantly lower recovery It was found that amitrol was hardly extracted in the organic solvent.
<실시예 3><Example 3>
상기 실시예 1 및 2와 같이 이소부틸 클로로포르메이트 50㎕을 반응시약으로 사용하고 실온에서 20분간 반응시켜서 얻은 용액을 2㎕ 취하여, 이 용액을 기체 크로마토그래피 또는 기체 크로마토그래피/질량 분석기에 주입하여 도 2에 나타낸 바와 같이 미량의 아미트롤과 내부표준물질의 피크의 크로마토그램을 얻었다. 도 3은 유도체화된 아미트롤의 질량스펙트럼을 나타내고 있는데, 유도체화된 각각의 분자량과 특성 토막이온이 잘 나타나 있다.50 μl of isobutyl chloroformate was used as a reaction reagent as in Examples 1 and 2, and 2 μl of a solution obtained by reacting at room temperature for 20 minutes was injected into a gas chromatography or a gas chromatography / mass spectrometer. As shown in Fig. 2, chromatograms of peaks of trace amounts of amitrol and internal standards were obtained. Figure 3 shows the mass spectrum of the derivatized amitrol, the molecular weight and characteristic fragment ions of each derivatized are well represented.
이와 같이 아미트롤의 농도를 결정하기 위해 사용된 기기는 휴렛-팩커드(Hewlett-Packard)사의 5973N 질량 분석기와 여기에 연결된 6890 플러스(plus) 기체 크로마토그래피이었다. 사용된 이온화 방법은 전자충격법(electron impact, EI)이며 이온원의 온도는 280℃이었다. 정량을 위하여 SIM(Selected Ion Monitoring) 모드가 사용되었으며 선택된 이온은 기준 피크(base peak)인 m/z 84가 사용되었다. 내부표준물질의 경우는 m/z 212가 사용되었다. 분석에 사용된 컬럼(column)은 길이가 25m, 내경이 0.25㎜, 필름 두께가 0.25㎛인 울트라(ultra) 2 컬럼을 사용하였고, 이동상 기체로는 헬륨을 사용하였으며, 흐름 속도는 0.8 ㎖/min이었다. 컬럼의 온도는 초기에 100℃에서 머무른 시간없이 10℃/min 속도로 300℃까지 승온하였다. 시료 주입구의 온도는 250℃이었으며 기체 크로마토그래프와 질량 분석기 연결 장치의 온도는 280℃이었다.The instrument used to determine the concentration of amitrol was a Hewlett-Packard 5973N mass spectrometer and 6890 plus gas chromatography connected thereto. The ionization method used was electron impact (EI) and the temperature of the ion source was 280 ° C. SIM (Selected Ion Monitoring) mode was used for quantification and m / z 84, the base peak, was used as the selected ion. For internal standards m / z 212 was used. The column used in the analysis was an ultra 2 column having a length of 25 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm, helium as the mobile phase gas, and a flow rate of 0.8 ml / min. It was. The temperature of the column was initially raised to 300 ° C. at a rate of 10 ° C./min without the time of staying at 100 ° C. initially. The temperature of the sample inlet was 250 ° C and the temperature of the gas chromatograph and the mass spectrometer connection device was 280 ° C.
<실시예 4><Example 4>
일부 정수장에서 채취한 시료를 10㎖ 취하여 내부표준물질인 피렌-d10 용액 (아세트산에틸 1㎖ 당 100㎍)을 10㎕ 첨가한 후 진공회전증발기를 이용하여 완전히 건조시키고 남아 있는 잔류물에 이소부틸 클로로포르메이트 50㎕를 첨가하여 실온에서 20분 동안 반응시켜 유도체화한 후, 질소 하에서 건조시켰다.Take 10 ml of sample from some water purification plant, add 10 µl of pyrene-d 10 solution (100 µg per 1 ml of ethyl acetate) as an internal standard, and completely dry it using a vacuum rotary evaporator. 50 µl of chloroformate was added, reacted for 20 minutes at room temperature, derivatized, and dried under nitrogen.
건조된 시료를 다시 아세트산에틸 100㎕로 용해시킨 후 2㎕를 직접 기체 크로마토그래피에 주입하여 분석하였다. 이러한 분석 방법을 통하여 아미트롤을 질량 분석기를 사용하여 정량하기 위한 검량곡선은 도 4에 나타낸 바와 같고 0.1 ∼ 100.0㎍/ℓ 농도 범위에서 좋은 직선성을 나타내었다.The dried sample was again dissolved in 100 µl of ethyl acetate, and 2 µl was directly injected into gas chromatography for analysis. The calibration curve for quantifying amitrol using this mass spectrometry through this analytical method is shown in FIG. 4 and showed good linearity in the concentration range of 0.1 to 100.0 µg / l.
상기 설명한 바와 같이, 본 발명은 물에 대한 용해도는 매우 높으나 일반적인 유기 용매에는 거의 녹지 않아 수질 시료로부터 유기 용매에 의한 추출이 어려운 아미트롤을 수질 시료 중에서 효과적으로 분석할 수 있도록 하며, 또한 본 발명은 기체 크로마토그래피 또는 기체 크로마토그래피/질량 분석기에 대해서 선택적이고 우수한 감도를 제공한다. 일반적으로 기체 크로마토그래피/질량 분석기로 직접 분석하기 어려운 아미노기를 이소부틸 클로로포르메이트와 반응시켜 유도체화함으로써 휘발성을 증가시키고 검출 감도를 향상되도록 하여, 물질 분석에 널리 사용되고 있는 기체 크로마토그래피/질량 분석기에 대하여 최적의 분석 조건을 제공한다. 따라서, 본 발명을 이용할 경우, 우수한 감도, 재현성 향상, 정확성, 분석 시간의 단축, 저비용 및 간편성 등을 만족시킬 수 있으며, 이로 인해 인체에 암을 유발할 수 있는 아미트롤을 수질 시료에서 정량적으로 분석할 수 있어 환경시료 특히 수질 시료 분석에 폭 넓게 응용할 수 있다.As described above, the present invention makes it possible to effectively analyze amitrol in the water sample, which has a very high solubility in water but is hardly soluble in a general organic solvent, which is difficult to extract from the water sample by the organic solvent. Provides selective and excellent sensitivity for chromatography or gas chromatography / mass spectrometry. In general, it is possible to increase the volatility and improve the detection sensitivity by reacting amino groups with isobutyl chloroformate, which are difficult to analyze directly by gas chromatography / mass spectrometer, to increase volatility and detection sensitivity. Provide optimal analysis conditions. Therefore, when using the present invention, it is possible to satisfy excellent sensitivity, improved reproducibility, accuracy, shortened analysis time, low cost and simplicity, and thus, quantitative analysis of amitrol, which can cause cancer in human body, can be quantitatively analyzed. It can be widely applied to the analysis of environmental samples, especially water samples.
도 1은 실시예 2에서 유기 용매로 추출하였을 경우와 유기 용매로 추출하지 않고 바로 농축하였을 경우의 아미트롤의 회수율을 나타낸 그래프.1 is a graph showing the recovery rate of amitrol when extracted with an organic solvent in Example 2 and immediately concentrated without extraction with an organic solvent.
도 2는 본 발명에 따라 기체 크로마토그래피/질량 분석기로 분석된 아미트롤의 크로마토그램.2 is a chromatogram of amitrol analyzed with a gas chromatography / mass spectrometer in accordance with the present invention.
도 3은 본 발명에 따라 유도체화된 아미트롤의 질량스펙트럼.3 is a mass spectrum of amitrol derivatized according to the present invention.
도 4는 본 발명에 따른 정량을 위한 검량곡선을 나타낸 그래프.Figure 4 is a graph showing the calibration curve for quantification according to the present invention.
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