1、中华人民共和国出入境检验检疫行业标准SN/T 1606-2005 进出口植物性产品中苯氧竣酸类除草剂残留量检验方法气相色谱法Inspection of phenoxy acid herbicides residues in products of plant origin for import and export-GC 2005-08-18发布060608000127 2006-02-01实施中华人民共和国发布国家质量监督检验检疫总局前言本标准的附录A、附录B和附录C为资料性附录。本标准由国家认证认可监督管理委员会提出并归口。本标准起草单位:中华人民共和国上海出入境检验检疫局。本标准主要起草
2、人z李波、郭德华、俞秋蓉、韩丽、王敏、王传现、王东辉、魏玉璜。本标准系首次发布的出入境检验检疫行业标准。SN/T 1606一2005I 1 范围进出口植物性产品中苯氧竣酸类除草剂残留量检验方法气相色谱法SN/T 1606-2005 本标准规定了进出口粮谷中麦草畏、2，4-滴丙酸、2，4-滴、2，4，5-三氯苯氧基丙酸、2，4，5-三氯苯氧基乙酸、2，4-滴丁酸残留量的抽样、制样和气相色谱-质谱测定方法。本标准适用于进出口小麦、大麦、大豆、油菜籽和大米中麦草畏、2，4-滴丙酸、2，4-滴、2，4，5-三氯苯氧基丙酸、2，4，5-三氯苯氧基乙酸、2，4-滴丁酸残留量的检验。2 抽样和制样2. 1
3、检瞌批以不超过4000袋为一检验批。同一检验批的商品应具有相同的特征，如包装、标记、产地、规格和等级等。2.2 抽样敢量按式(1)确定抽样数量。式中zN一一全批袋数;a一一抽样袋数。注:a值取整数，小数部分向前进位为整数。2.3 抽样工具a= IN ( 1 ) 2.3.1 金属单管取样器:不锈钢管，全长55cm(包括手柄)，直径1.5 cm，沟槽长度应超过袋对角线长度的一半。2.3.2 取样铲。2.3.3 分样板。2.3.4 样品筒(袋):可密封。2.3.5 分样布或适用铺垫物。2.4 抽样方法2.4. 1 倒包抽样从堆垛的各部位随机抽取2.2规定的应抽样件数的10%(每批一般不少于3袋)，将
4、袋口缝线全部拆开，平置于分样布或其他洁净的铺垫物上，双手紧握袋底两角，提起约成45。倾角，倒拖约1m，使袋内货物全部倒出。查看袋内和袋间品质是否均匀。确认情况正常后，用取样铲随机在各部位抽取样品，立即将样品倒入盛样器内。每袋抽取样品数量应基本一致。2.4.2 袋内抽样按2.2规定的应抽样袋数的90%，在堆垛四周上、中、下各层以曲线形走向随机抽取。将取样器(2.3. 1)管槽朝下，从每袋一角依斜对角方向插入袋内，然后将管槽旋转朝上，抽出取样器，立即将样品倒入盛样器内。每袋抽取样品数量应与2.4.1基本一致。2.4.3 大样细分集中倒包抽样和袋内抽样所取全部样品，倒于分样布上，用分样板按四分法缩分
5、出样品不少于SN/T 1606-2005 2峙，盛于样品筒内，加封后，标明标记并及时送交实验室。2.5 试样制备将样品按四分法缩分出约1峙，全部磨碎并通过20目筛，混匀，均分成两份试样，装入洁净的容器内，密封，标明标记。2.6 试样保存将试样于一5-C以下避光保存。3 测定方法3. 1 方法提要试样中的苯氧竣酸类除草剂用丙酣-乙酷在pH值为2的酸性条件下提取，提取液经氢氧化饵榕液净化除去脂溶性杂质后，再调pH值至小于2后，用乙酷提取，浓缩，重氮甲烧衍生化，用配有多级质量选择检测器的气相色谱仪测定，外标法定量。3.2 试剂和材料除另有规定外，所用试剂均为分析纯，水为蒸锢水。3.2.1 丙酣。3.
6、2.2 无水乙酿。3.2.3 正己烧。3.2.4 异辛烧。3.2.5 甲醇。3.2.6 浓盐酸。3.2.7 浓硫酸。3.2.8 浓磷酸。3.2.9 酸化的无水硫酸铀:650.C灼烧4h，在干燥器中冷却至室温。用无水乙酷浸没100g无水硫酸纳固体表面，加0.1mL浓硫酸并充分混合。通风橱中挥去乙醋。酸化测试z将1g硫酸铀与5mL蒸馆水棍合，pH小于4。使用前130c活化4h。3.2.10 磷酸缓冲溶液(0.1mol!L):称取12g磷酸二氢铀溶解于1L蒸馆水中，用磷酸调节溶液pH到2.5.3.2. 11 三甲硅基重氮甲烧溶液:2mol!L，市售。3.2.12 37%氢氧化饵水溶液z称取37g氢氧
7、化饵溶解于100mL蒸馆水中。3.2. 13 碱水溶液:37%氢氧化饵与蒸馆水的体积比0+2)。3.2. 14 硫酸水溶液z硫酸-水0+3)，储存于4.C冰箱中。3.2. 15 麦草畏标准品E纯度二三99%。3.2.16 2，4-滴丙酸标准品:纯度二三99%。3.2.17 2，令滴标准品z纯度注99%。3.2.18 2，4，5-三氯苯氧基乙酸标准品:纯度;?;99%。3.2.19 2，4-滴丁酸标准品z纯度二三99%。3. 2. 20 2 ，4，5-三氯苯氧基丙酸标准品z纯度二三97%。3.2.21 麦草畏、2，4-滴丙酸、2，4-滴、2，4，5-三氯苯氧基丙酸、2.4，5-三氯苯氧基乙酸、2
8、，4-滴丁酸标准储备液z各准确称取0.0100g标准品，分别用甲醇榕解定容至100mL，溶液浓度为100g/mL，存放于4C冰箱中。3.2.22 麦草畏、2，4-滴丙酸、2，今滴、2，4，5-三氯苯氧基丙酸、2.4，5-三氯苯氧基乙酸、2，4-滴丁酸标准混合溶液z根据需要用甲醇稀释成适用浓度的标准温合溶液。3.3 仪器和设备3.3. 1 气相色谱仪z配有多级质量选择检测器(MSMS)。3.3.2 振荡器。3.3.3 涡旋器。3.3.4 离心机:5000 r/mn。3.3.5 旋转蒸发器。3.3.6 氮吹仪。3.3.7 塑料离心瓶:150 mL。3.3.8 分液漏斗:125mL、500mL。3.
9、3.9 容量瓶:50mL、100mL。3.3.10 离心管:50mL、100mL。3.3. 11 衍生瓶:4mL。3.3. 12 浓缩瓶:150mL。SNjT 1606-2005 3.3. 13 酸化的无水硫酸铀干燥柱:80 mm X 20 mm (内径)筒形漏斗，底部垫少许脱脂棉，再装入50 mm高的酸化无水硫酸锅。3.3.14 锥形瓶:500mL，具塞。3.3. 15 微量注射器:10L。3.4 测定步骤3.4. 1 提取准确称取10.0g磨碎的均匀试样于150mL塑料离心瓶中，加入30mL磷酸缓冲溶液，混匀，用浓盐酸调节pH至2，加入10mL丙圃，振荡20min，再加人40mL无水乙酶，
10、振荡20min，于3500r/min离心5mn。将上层潜液转移至装有200mL蒸锢水的500mL分液漏斗中，残渣再分别用10mL丙酣和40mL无水乙酷重复提取两次，合井上层溶液于上述分液漏斗中，轻缓振摇1mn，静置分层，收集乙瞠层，水层再用25mL无水乙酷重复提取一次，合并乙酷层，于300C水浴下减压浓缩至约10mL. 3.4.2 净化将试样榕液移入50mL离心管中，加入15mL碱水溶液，充分混匀2min，于1500 r/mn离心10 min，移取水相，乙酷相再用15mL碱水溶液重复提取两次，合并水相，若试样含油脂量高(如大豆、油菜籽等)，附加10mL无水乙醋于水相中，充分混匀2min，于15
11、00 r/min离心10min，弃酷层。水相转移至125mL分液漏斗中，小心用硫酸水溶液调节pH小于2，冷却后，加入40mL无水乙醋，振摇2min，静置分层，收集乙酷层。水层再用20mL无水乙酷重复提取两次，合并乙酷层。经酸化的无水硫酸饷干燥柱脱水，收集于含10g酸化的无水硫酸铀的锥形瓶中，不时振摇，2h后，倾出乙酷相于30.C水浴下减压浓缩至近干。3.4.3 衍生化将残渣用无水乙酷溶解并转移至4mL衍生瓶中，在30.C水浴下用平缓氯气流吹干，加人200L异辛烧、200L甲醇、400L三甲硅基重氮甲皖溶液，涡旋握匀，70.C下保持10min。冷至室温后，用平缓氮气流吹干，用正己烧定容至1mL，
12、过0.45m微孔滤膜，滤液供气相色谱-质谱测定。标准工作溶液同步进行衍生测定。3.4.4 测定3.4.4. 1 气相色谱质谱条件a) 色谱柱:CP-SIL8LOW BLEED/MS型毛细管柱，60mXO. 25 mm(内径)X 0.25m(膜厚), 或相当者;b) 载气z氮气，纯度二三99.999%，流速1.2 mL/mn; c) 柱温:70.C，保持1min，以10OC/mn的速度升温到190.C，保持2min，再以50C/mn的速度升SN/T 1606一2005温到250.C，保持10min; d) 进样口温度:260.C;e) 进样方式z无分流进样，0.75min后开阔pf) 进样量:1
13、L; g) 离子阱温度:150.C ; h) 传输线温度:200.C;i) 灯丝电流:80A;j) 溶剂延迟:14.20 min; k) MS/MS监测:六种苯氧竣酸类除草剂根据保留时间分为六个时段检测，每种化合物的分析时段、保留时间、母离子、定性子离子、定量子离子、质量扫描范围、碰撞电压值等参数，参见附录A。3.4.4.2 气相色谱质谱测定根据样液中被测组分含量，选定浓度相近的标准工作洛液。标准工作溶液和样液中除草剂的响应值均应在仪器检测的线性酒围内。对标准工作溶液与样液等体积参插进样测定。在上述色谱条件下，除草剂标准品对应的衍生物选择离子色谱图参见附录B。定性测定，样液如果检出的色谱峰的保
14、留时间与标准榕液中某种除草剂相一致，并且所选择的子离子均出现，而且之间的丰度比也相一致，则可判定试样中含有该种除草剂。除草剂标准品对应的衍生物的质谱图参见附录C。3.4.5 空白试验除不加试样外，均按上述测定步骤进行。3.4.6 结果计算和表述用色谱数据处理机或按式(2)计算样品中各种苯氧竣酸类除草剂残留含量，计算结果应将空白值扣除。式中:X=AXcXV As Xm X一一试样中各种苯氧竣酸类除草剂的残留含量，单位为毫克每千克(mg/kg); A一一样液中各种苯氧竣酸类除草剂的峰面积FAs-标准工作溶液中各种苯氧竣酸类除草剂的峰面积;c一一标准工作溶液中各种苯氧竣酸类除草剂的浓度，单位为微克每
15、毫升(g/mL); V一一一】样液最终定容体积，单位为毫升(mL); m一一试样量，单位为克(g)。4 测定低限、回收率4. 1 测定低限. ( 2 ) 本方法对下列除草剂的测定低限为z麦草畏0.025mg/kg; 2，4-滴丙酸0.05mg/kg; 2, 4-滴0.05 mg/kg;2，4，5-三氯苯氧基丙酸0.05mg/kg;2，4，5-三氯苯氧基乙酸O.05 mg/kg; 2，4-滴丁酸0.05 mg/kg。4.2 回收率在小麦、大麦、大豆、油菜籽、大米中麦草畏，2，4-滴、2，4-滴丙酸、2，4-滴丁酸、2，4，5-三氯苯氧基丙酸和2，4，5-三氯苯氧基乙酸的添加浓度及其回收率实验数据
16、见表1。4 SN/T 1606一2005表1实验数据表除草剂名称添加浓度/Cmg/kg)回收率范围麦草畏0.025 90%99% O. 10 101%103% 0.25 96%104% 2，4-滴丙酸0.05 80%90% 0.20 85%-95% 0.50 90%100% 2，4-滴0.05 78%90% 0.20 80%95% 0.50 88%96% 2，4，5-三氯苯氧基丙酸0.05 72%84% 0.20 75%85% 0.50 80%92% 2，4，5-三氯苯氧基乙酸0.05 82%92% 0.20 92%102% 0.50 90%100% 2，4-滴丁酸0.05 70%80% 0.
17、20 70%80% 0.50 80%84% 5 SN/T 1606-2005 附录A(资料性附录)GC-MS/MS测定六种除草剂的检测参数表A.1分析时段/质量扫描保留时间/二级质谱二级质谱监定量离子化合物CID电压母离子测子离子mm 范围/amumm (m/z) (m/z) (m/z) 188 麦草畏14.20-15.50 0.96 45-250 14.518 203 175 188 147 2，4-滴丙酸15.50-16.10 1. 05 45-300 162 15.913 248 189 162 213 156 2，4-滴16.10-17.00 0.65 45-250 16.377 19
18、9 125 156 141 2，4，5-三196 氯苯氧17.00-18.60 0.55 45-300 18.308 282 247 196 基丙酸223 2，4，5-三190 氯苯氧18.60-19.30 1. 05 45-300 18.932 233 159 190 基乙酸218 2，4晴滴59 19.30-20.60 0.65 45-200 20. 104 101 59 丁酸101 6 附录B(资料性附景)标准晶衍生物选择离子色谱固kCounts 500 400 300 200 100 。13 14 1一一麦草畏14.518min; 2一-2，4-滴丙酸15.913min; 3一-2，4
19、-滴16.377min; 15 4一一2，4，5-三氯苯氧基丙酸18.308min; 5一-2，4，5-三氯苯氧基乙酸18.932min; 6一-2，4-滴丁酸20.104mino 3 16 17 4 18 19 圄B.1六种苯氧援酸类除草剂标准晶衍生物的选择离子色谱圈SN/T 1606-2005 6 20 mmet臼7 SN/T 1606-2005 100 50 。50 70 100% 75% 50% 25% 68 0% 50 8 CI 97 附录C(资料性附录)标准晶质谱固Cl 203 188 234 90 110 130 150 170 190 210 230 250 a)级质谱图188
20、 175 147 93 254 281 296 100 150 200 250 m/z b)二级质谱图固C.1麦草畏标准晶衍生物的质谱圈100 50 100% 75% 50% ,i 25% SN/T 1606-2005 59 H 一248 162 40 50 60 70 80 90 100 110 120 130 140 150 160170 180 190 200210 220 230240 250 260 a)一级质谱图162 189 213 83 92 103 125 1.1, 25 100 150 200 250 m Iz b)二级质谱图固C.22，4-滴丙酸标准晶衍生物的质谱圈9 S
21、N/T 1606一2005199 100 OV人。/50 234 111 161 176 146 。60 70 90 110 130 160 170 190 210 230 250 a)一级质谱图100% 156 75% 50% 125 25% 200 I .1, 234 247 0% 50 1 150 2 m/z b)二级质谱图固C.32，4-滴标准晶衍生物的质晴圆10 100 50 60 100% 75%: 50% 25% 91 . . 100 固C.4、丫Cl汇87 196 SN/T 1606-2005 282 223 80 100 120 140 160 180 200 220 240
22、 260 280 300 a)一级质谱图196 247 223 283 132 159 265 299 . 150 200 250 m/z b)二级质谱图2.4.5-三氯苯氧基芮酸标准晶衍生物的质谱固11 SN/T 1606-2005 100 50 。100% 75% 50% 25% 0% 12 233 A/ Cl Cl 268 Cl 209 181 50 70 90 110 130 150 170 190 210 230 250 270 a)一级质谱图190 159 218 235 270 100 150 200 250 m /z b)二级质谱图固C.52，4，5-三氯苯氧基Z酸标准晶衍生物
23、的质谱固SN/T 1606-2005 101 100 59 人/50 162 231 。50 70 90 110 130 150 170 190 210 a)一级质谱图100% 59 75% 50% 25% 102 124 152 204 215 226 248 t 100 150 200 250 m /Z b)二级质谱囱固C.62，4-滴丁酸标准晶衍生物的质谐圄13 SN/T 1606-2005 Foreword Annex A , annex B and annex C of this standard is an informative annex. This standard was
24、proposed band is under the charge of the Certification and Accreditation Admin istration of the Peoples Republic of China. This standard was drafted by Shanghai Entry-Exit Inspection and Quarantine Bureau of the Peoples Republic of China. The main drafters of this standard are Li Bo , Guo Dehua , Yu
25、 Qiurong , Han Li, Wang Min , Wang Chuanxian , Wang Donghui and Wei Yupu. This standard is a professional standard of ent叩-exitinspection and quarantine promulgated for the first time. Note:this English version ,a translation from the Chinese text,is solely for guidance. 14 SN/T 1606-2005 Inspection
26、 of phenoxy acid herbicides residues in plant origin products for import and export-GC 1 Scope This standard specifies the methods of sampling.回mplepreparation and determination of 2. 4-0. 2.4-DP. 2.今DB.2.4.5-TP. 2.4.5-T and Dicamba residues by GC/MS in plant origin products. This standard is applic
27、able to the determination of 2.4-D.2.4-DP.2.4-DB. 2.4.5-TP. 2.4.5-T and Oi camba residues in wheat.国rley.soybean. coleseed and rice for import and export. 2 Sampling and sample preparation 2. 1 Inspection lot The quantity of an inspection lot should not exceed 4 000 packages. The characteristics of
28、the cargo within the same inspection lot. such as packing. mark. origin. spec ification and grade. should be the same. 2. 2 Quantity of回mpletaken Sampling taken is according to the formula (1). a= .f百Where , N-total number of bags in an inspection lot; a-number of bags to be taken. . ( 1 ) Note: If
29、value a is with decimal , round off the decimal part. which is added as unity to the int啕ralpart of 8. 2. 3 Sampling tools 2. 3. 1 Metallic sampler: stainless steel; length (including handle) : 55 cm; diameter: 1. 5 cm; groove length: longer than half the diagonal length of the bag. 2.3.2 Sampling s
30、hovel 2. 3. 3 Plate for quartering 2. 3. 4 Sample container: Can or bag. which臼nbe sealed. 2.3.5 Cloth (or other suitable materiaDsheet: For sample dividing (quartering) 15 SN/T 1606-2005 2. 4 5ampling procedure 2.4. 1 Sampling by empting out Draw 10 percent of the number of bags specified in 2.2 (n
31、ot less than three bags) at any part of the pile at random . Unseal and open the bag , and place it on the sampling cloth sheet (or other clean sheeO , Grasp tight two corners of the bags bottom and raise up t an angle of 45 degree, tug back ward for ca 1m until all contents of the bag is emptied ou
32、t. Check whether the quality of goods is uni form within and between the bags. After confirming the goods are in normal condition , scoop up the sample from different parts of the out-poured content at random, and promptly place in a clean sam ple container. The quantity of the sample drawn from eac
33、h bag should be basically the same. 2. 4. 2 Sampling from nside the bags Draw the samples from 90 percent of the number of bags specified in 2.2 (bdeducting the number of bags drawn in 2.4. 1. 1). Along the sine wave of the pile, draw the samples from the bags of the upper, middle and lower parts ar
34、ound the pile at random. Insert the sampler, with its gr，ve facing downward, diagonallinto each bag , then turn the sampler by 180 degree, draw out the sampler and promptly pour the sample into a container. The quantity of the sample drawn from each bag should be basically the same as in 2. 4. 1. 2.
35、 4. 3 Reduction of gross回mplePour all of samples on a clean sheet, reduce to not less than 2 kg with a plate by quartering. Place in a sample container, seal , label and sent to the laboratory in time. 2. 5 Preparation of test sample Reduce the sample to ca 1 kg by quartering, grind thoroughland let
36、 pass through a 20 mesh sieve, mix thoroughly and divide into 2 equal portions. Each portion is placed in a clean container as the test sample, seal and label. 2. 6 Storage of test回mpleThe test samples should be stored below一5C and kept awafrom light. 3 Method of determination 3. 1 Principle Phenoxy
37、-acid Herbicides in the test sample are extracted with acetone-ether under the condition of acid with a pH = 2. After cleaned up to deprivate the liposolubility impurity through sodium hydrox ide solution, adjust the pH to below 2 , then extracted with ether, concentrated , and derivatizated bdiazom
38、ethane. Determination made by means of gas chromatography equipped with multiple mass selective detector, using external standard method to quantitation. 16 SN/T 1606-2005 3.2 Re暗entsand materials Unless otherwise specified , the reagents should be analtically pure; Water is distilled water. 3.2.1 A
39、cetone. 3.2.2 Anhydrous ether. 3.2.3 Hexane. 3.2.4 Isooctane. 3.2.5 Methanol. 3.2.6 Concentrated hydrochloride acid. 3.2.7 Concentrated sulfuric acid. 3.2.8 Concentrated phosphoric acid. 3.2.9 Acidified anhydrous sodium sulfate: Heated at 650t for 4 hours, then cooled to room tem perature. 100 9 anh
40、ydrous sodium sulfate are covered the solid by anhydrous ether; then add O. 1 mL of concentrated sulfuric acid and mix thoroughly. Remove the ether in the fume hood. Acidify test: Mix Ig of the resulting solid with 5 mL of distilled water and measure the pH of the mixture. The pH must be below 4. Ac
41、tivated 4 hours at 1300C before using. 3.2.10 Phosphate buffer (0. 1 mol/U: weight 12 9 sodium phosphate (NaH2P04) and dissolve in 1.0 L-distilled water. Add phosphoric acid to adjust the pH to 2.5. 3.2.11 Diazomethane: 2 mol/L,commercially. 3.2.12 37% potassium hydroxide solution: weight 37 9 potas
42、sium hydroxide and dissolve in 100 mL-distilled water. 3.2.13 Alkaline (basic) solution: the volume ratio of 37% potassium hydroxde and dstilled water s (1 +2). 3.2.14 Sulfate acd soluton: the volume rato s sulfate acd -water( 1 + 3) , stored n refrgerator at 40C. 3.2.15 Dcamba standard: Purty99%. 3
43、.2.16 2,4-DP standard: Purty注99%.17 SN/T 1606一20053.2. 17 2.4-0 standard: Purity二99%.3.2.18 2.4.5-T standard: Purity二三99%.3.2.19 2.4-0Bstandard: Purity注99%.3.2.20 2.4.5-TP standard: Purity97%. 3.2.21 Oicamba; 2.4-0P; 2.4-0. 2.4.5-TP; 2.4.5-T; 2.4-0B standard stock solution: accurately weighing appro
44、ximate 0.0100 9 standard and volume to 100 mL with methanol. The solution is 100 g/mL in concentration and stored in the refrigerator at 4 t . 3.2.22 Oicamba; 2.4-0P; 2.4-0; 2.4.5-TP; 2.4.5-T; 2.4-0Bstandardmixturesolution: Oilutethe standard stock solution with methanol to the required concentratio
45、n as the standard working solu tion. 3. 3 Apparatus and明uipment3.3.1 Gas chromatograph: equipped with multiple select ion mass detector (MS/MS). 3.3.2 Wrist shaker. 3.3.3 High speed homogenizer. 3.3.4 Centrifuge: 5000 r/min. 3.3.5 Rotary evaporator. 3.3.6 Nitrogen evaporator 3.3.7 Centrifuge bottle-
46、Pasteur: 150 mL. 3.3.8 Separatory funnel: 125 mL. 500 mL. 3.3.9 Volumetric flasks: 50 mL. 100 mL. 3.3.10 Centrifuge tube: 50 mL, 100 mL. 3.3.11 Oerivation bottle: 4 mL. 3.3.12 Concentration bottle: 150 mL. 3.3.13 Acidified anhdrous sodium sulfate drying column:80 mm x 20 mm ( internal diameter ) 18
47、SN/T 1606-2005 funnel. A little absorbent cotton cover the bottorn and add acidified anhydrous sodium sulfate with 50 mm high. 3. 3. 14 Erlenmeer flask: 500 mL , with stopcock. 3.3.15 Micro-syringe: 10L. 3. 4 Procedure 3. 4. 1 Extraction Accurately weigh 10.0 9 test sample ground finely into a 150 m
48、L plastic Centrifuge bottle, add 30 mL phosphate buffer, mixing uniformly, and adjust the pH to 2 with hydrochloric acid. Add 10 mL ace tone and shock for 20 min , then add 40 mL ether again and shock for 20 min. Centrifuge for 5 min un der 3500 r/min. After transfer the upper solution to a 500 mL S
49、eparatory funnel contained 200 mL distilled water, residents are repeat extracted for 2 times with 10mL acetone and 40 mL anhydrous ether. Combine the upper layer solution to the same 500 mL Separatorfunnel， gently mix for 1 min and set aside for separating. Collect the ether layer, and the aqueous fraction is re-extracted one time using 25 mL of anhdrous ether. Combine ether extract and concentr
