薄层色谱法检测果蔬中仲丁胺残留量技术研究
摘要
仲丁胺(2-aminobutane，简称2-AB)是一种保护性杀菌剂，对多种真菌有抗菌活性，广泛应用于柑桔、苹果、梨、桃、香蕉、马铃薯、青椒等果品蔬菜贮藏期的防腐保鲜。由于2-AB不能用分光光度计进行直接检测，同时果蔬样品中含有其他低级脂肪胺干扰测定。所以针对上述问题本文分别采取待测物衍生和薄层色谱分离的办法，通过对衍生条件与薄层色谱分离条件以及分离后定量分析方法的筛选，确定了薄层色谱法检测果品蔬菜中仲丁胺残留量的操作过程如下：取一定量样品匀浆→加入75ml lmol／LCaCl_2溶液，10g MgO加150ml蒸馏水→水蒸汽蒸馏→用装有10ml 0.15mol／L H_2SO_4溶液的量筒接收馏出液约80ml→用0.3 mol／L NaOH溶液将蒸馏液调到pH7→用蒸馏水定容至100ml→吸取样品溶液10 ml于125ml梨形分液漏斗中→分别用15ml四氯化碳洗涤三次→转至50ml具塞锥形瓶中→加入5ml硼酸盐缓冲液和2ml DNFB试剂置60℃水浴反应30min→加入2ml 2mol／L NaOH溶液继续恒温30min→冰浴冷却→用10ml环己烷萃取，并弃去水层→再用15ml 0.05mol／L Na_2CO_3溶液洗涤共三次→加入2g无水Na_2SO_4脱水→旋转蒸发至干→0.2ml苯溶解→将50μl上述溶液点于硅胶薄层板→用正己烷-乙醚(70：30)展开→以三氯甲烷洗脱斑点并定容至5ml→于332nm处测吸光度。为保持一致的操作条件，系列标准溶液采用同样的衍生、薄层色谱分离和定量分析方法，绘制标准曲线。该检测方法的综合评价结果表明，其线性范围为1.25～25μg，回归方程为Y=0.0121X-0.0071，相关系数为R~2=0.9945，最低检出量为1μg，对青椒、蒜苔、柑桔、葡萄检测的回收率分别为101.2％、89.2％、88.5％、85.7％，RSD分别为7.9％、5.4％、7.7％、4.1％。
2-AB is a kind of protective fungicide that is effective to a range of fungi. It is a preservative that is widely used in the storage of the orange, apple, pear, peach, banana, potato, green pepper etc. It is difficult to detect the residue of 2-AB by UV detector directly. The other low molecular weight aliphatic amines in some fruit and vegetables will interfere the detecting. To get an accurate result, it is necessary to convert 2-AB into its derivative by reaction between 2-AB and DNFB, and to separate 2-AB derivative from other derivatives by thin layer chromatography. In this paper, the conditions of deriving, TLC and quantitative analysis were studied. The selected procedures for detection of 2-AB residue in fruit and vegetables are as follows: take some slurry → add 75ml 1 mol/L CaCl2, 10g MgO and 150ml distilled water → distill with vapor → receive 80ml distillate fraction with 10ml 0.15 mol/L H2SO4 → adjust pH to 7 with 0.3 mol/L NaOH → dilute to 100ml with distilled water → get sample solution→ tak
e 10ml sample solution into 50ml separatory funnel → extract with 15ml carbon tetrachloride for three times → transfer into 50ml conical flask with plug → add 5ml borate buffer liquid and 2ml DNFB, keep in water bath at 60 ℃ for 30 min → add 2ml 2mol/L NaOH and keep for another 30 min →cool with ice bath → extract with cyclohexane and discard water layer → wash with 15ml 0.05mol/L Na2CO3 for three times→ add 2g Na2SO4 without water to dehydrate → vaporize to dryness → resolve in 0.2ml benzene → spot 50ul solution on thin layer → spread with hexane: ether (70:30) → wash the 2-AB spot with chloroform and to make up to 5ml → detect under 332nm. A series of 2- AB standard solution are derived, separated and quantitative analyzed at same conditions with same methods. Using this method, the content of 2-AB has
wonderful liner relationship with its absorbance value, the regress equation is Y=0.0121X-0.0071, R2=0.9945, the liner range is 1.25~25ng, the lowest detecting amount on thin layer is l(ig. The detection of some fruits and vegetables shows that the recoverage for pepper, garlic bolt, orange and grape are 101.2%, 88.5%, 85.7% and 89.2%, respectively, and the RSD for them are 7.9%, 5.4%, 7.7% and 4.1%, respectively.
2-AB is a kind of protective fungicide that is effective to a range of fungi. It is a preservative that is widely used in the storage of the orange, apple, pear, peach, banana, potato, green pepper etc. It is difficult to detect the residue of 2-AB by UV detector directly. The other low molecular weight aliphatic amines in some fruit and vegetables will interfere the detecting. To get an accurate result, it is necessary to convert 2-AB into its derivative by reaction between 2-AB and DNFB, and to separate 2-AB derivative from other derivatives by thin layer chromatography. In this paper, the conditions of deriving, TLC and quantitative analysis were studied. The selected procedures for detection of 2-AB residue in fruit and vegetables are as follows: take some slurry → add 75ml 1 mol/L CaCl2, 10g MgO and 150ml distilled water → distill with vapor → receive 80ml distillate fraction with 10ml 0.15 mol/L H2SO4 → adjust pH to 7 with 0.3 mol/L NaOH → dilute to 100ml with distilled water → get sample solution→ tak
e 10ml sample solution into 50ml separatory funnel → extract with 15ml carbon tetrachloride for three times → transfer into 50ml conical flask with plug → add 5ml borate buffer liquid and 2ml DNFB, keep in water bath at 60 ℃ for 30 min → add 2ml 2mol/L NaOH and keep for another 30 min →cool with ice bath → extract with cyclohexane and discard water layer → wash with 15ml 0.05mol/L Na2CO3 for three times→ add 2g Na2SO4 without water to dehydrate → vaporize to dryness → resolve in 0.2ml benzene → spot 50ul solution on thin layer → spread with hexane: ether (70:30) → wash the 2-AB spot with chloroform and to make up to 5ml → detect under 332nm. A series of 2- AB standard solution are derived, separated and quantitative analyzed at same conditions with same methods. Using this method, the content of 2-AB has
wonderful liner relationship with its absorbance value, the regress equation is Y=0.0121X-0.0071, R2=0.9945, the liner range is 1.25~25ng, the lowest detecting amount on thin layer is l(ig. The detection of some fruits and vegetables shows that the recoverage for pepper, garlic bolt, orange and grape are 101.2%, 88.5%, 85.7% and 89.2%, respectively, and the RSD for them are 7.9%, 5.4%, 7.7% and 4.1%, respectively.
引文
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3．陈秀华，陶辛秋．1988．仲丁胺在果蔬贮藏中的防腐作用．农业科技通讯．(11)：30
4．陈悦娇，马应丹，周妍芳．2002．薄层层析-紫外分光光度法测定白果中的β—胡萝卜素．仲恺农业技术学院学报．15(2)：41-44
5．陈治远．1995．仲丁胺系列保鲜剂的研究与应用．山西果树．(4)：13-14
6．陈治远．1996．仲丁胺系列柑桔保鲜剂的应用技术．中国农业科学通报．12(4)：50-51
7．崔同，傅承光．1995(a)．对肽内酰胺苯磺酰氯柱前衍生高效液相法检测植物体内痕量多胺．色谱．13(2)：117-119
8．崔同，傅承光．1995(b)．对肽内酰胺苯磺酰氯柱前衍生HPLC法测定仲丁胺残留量．分析测试学报．14(5)：53-55
9．崔同，傅承光．1996．对肽内酰胺苯磺酰氯与胺类化合物的衍生反应．应用化学．13(5)：97-99
10．崔同，李晓丽，傅承光，王燕桓．1997．对肽内酰胺苯磺酰氯的合成及性质研究．化学试剂．19(6)：363-364
11．丁雅韵，谢孟峡，邓志威．2001．丹磺酰氯作为氨基酸柱前衍生试剂衍生条件的研究．北京师范大学学报．37(4)：526
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13．邓伯勋．2002．园艺产品贮藏运销学．北京：中国农业出版社：137
14．董慧如，金花．2002．薄层色谱法在定量分析中的应用．理化检验-化学分册．38(5)：265-270
15．范继善．1993．实用食品添加剂．天津科学技术出版社：24
16．何丽一．2000．平面色谱方法及应用．北京：化学工业出版社
17．侯保林．1998．果蔬病害防治技术．北京：中国农业出版社：318
18．胡鸿，赵华，吴肇志，陶辛秋．1993．甜椒贮藏病害及其防腐措施的研究．中国蔬菜．(6)：12-14
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20．靳敏，夏玉宇．2003．食品检验技术．北京：化学工业出版社
21．李秉超，明霞，李晓波．2000．有机化学．北京：中国农业出版社：234
22．李向荣，方晓．1997．绿色蔬菜中V-_(kl)的薄层-紫外分度光度测定．食品科学．18(8)：47-49
23．刘勤冬，侯玉娥．1998．薄层．紫外法测定水稻种衣剂中多菌灵和福美双含量．安徽化工．95(5)：38-39
24．唐福军，张成林，孙太凡．2001．多菌灵薄层层析-紫外分光光度法的研究．黑龙江八一农垦大学学报．13(1)：82-84
25．王宏亮．1998．薄层层析法测定饲料中黄曲霉毒素B_1方法的改进．粮食与饲料工业．(1)：40-42
26．王振文，董霞．1985．仲丁胺及其衍生物对于多种水果、蔬菜贮藏期防腐保鲜效果的研究．河北农业大学学报．8(3)：23-35
27．吴潇．2001．薄层色谱法在氯霉素片含量分析中的应用．药物分析杂志．21(5)：368
28．熊小敏．1 997．薄层-溴化法测定福美双和福美锌含量．农资科技．(5)：23-24
29．杨文熙，陈辉平，张瑞敏，范志斌．1985．低级脂肪胺的气相色谱分析．河北农业大学学报．8(3)：72-81
30．叶凤阁，张忠源．1991．仲丁胺抑菌作用及其机理．北方园艺．(2)：15-18
31．俞加林，赵敏，黄湘倚．1991．国内水果保鲜剂研究进展．北方园艺．69(1)：35-37
32．张胜强，许清．1995．TLC-UV法测定小鼠体内的安定．中国药科大学学报．26(1)：20-22
33．张晓平，杨国林，何庆，米沙．2002．柱前衍生化反相高效液相色谱法测定N-甘氨酰
-L-谷氨酰胺—水合物含量．药物分析杂志．22(2)：89-91
34．张忆华，杨家顺．2001．食品中的农药残留及一些对策．食品发酵工业．27(7)：68-71
35．张志云，田勇，宋壮兴，冯晓元．1996．几种防腐剂对梨贮藏保鲜效果的实验．北方果树．(3)：13-15
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2．陈婷，朱丹妮．1995．薄层色谱-紫外分光光度法测定柴胡疏肝散中柴胡皂甙a(d)的含量．中国药科大学学报．26(6)：353-355
3．陈秀华，陶辛秋．1988．仲丁胺在果蔬贮藏中的防腐作用．农业科技通讯．(11)：30
4．陈悦娇，马应丹，周妍芳．2002．薄层层析-紫外分光光度法测定白果中的β—胡萝卜素．仲恺农业技术学院学报．15(2)：41-44
5．陈治远．1995．仲丁胺系列保鲜剂的研究与应用．山西果树．(4)：13-14
6．陈治远．1996．仲丁胺系列柑桔保鲜剂的应用技术．中国农业科学通报．12(4)：50-51
7．崔同，傅承光．1995(a)．对肽内酰胺苯磺酰氯柱前衍生高效液相法检测植物体内痕量多胺．色谱．13(2)：117-119
8．崔同，傅承光．1995(b)．对肽内酰胺苯磺酰氯柱前衍生HPLC法测定仲丁胺残留量．分析测试学报．14(5)：53-55
9．崔同，傅承光．1996．对肽内酰胺苯磺酰氯与胺类化合物的衍生反应．应用化学．13(5)：97-99
10．崔同，李晓丽，傅承光，王燕桓．1997．对肽内酰胺苯磺酰氯的合成及性质研究．化学试剂．19(6)：363-364
11．丁雅韵，谢孟峡，邓志威．2001．丹磺酰氯作为氨基酸柱前衍生试剂衍生条件的研究．北京师范大学学报．37(4)：526
12．邓伯勋．1992．柑桔果品贮运保鲜技术．北京：中国农业科技出版社：91-92
13．邓伯勋．2002．园艺产品贮藏运销学．北京：中国农业出版社：137
14．董慧如，金花．2002．薄层色谱法在定量分析中的应用．理化检验-化学分册．38(5)：265-270
15．范继善．1993．实用食品添加剂．天津科学技术出版社：24
16．何丽一．2000．平面色谱方法及应用．北京：化学工业出版社
17．侯保林．1998．果蔬病害防治技术．北京：中国农业出版社：318
18．胡鸿，赵华，吴肇志，陶辛秋．1993．甜椒贮藏病害及其防腐措施的研究．中国蔬菜．(6)：12-14
19．江苏省畜牧兽医学校．1996．果蔬贮藏保鲜加工大全．北京：中国农业技术出版社：71
20．靳敏，夏玉宇．2003．食品检验技术．北京：化学工业出版社
21．李秉超，明霞，李晓波．2000．有机化学．北京：中国农业出版社：234
22．李向荣，方晓．1997．绿色蔬菜中V-_(kl)的薄层-紫外分度光度测定．食品科学．18(8)：47-49
23．刘勤冬，侯玉娥．1998．薄层．紫外法测定水稻种衣剂中多菌灵和福美双含量．安徽化工．95(5)：38-39
24．唐福军，张成林，孙太凡．2001．多菌灵薄层层析-紫外分光光度法的研究．黑龙江八一农垦大学学报．13(1)：82-84
25．王宏亮．1998．薄层层析法测定饲料中黄曲霉毒素B_1方法的改进．粮食与饲料工业．(1)：40-42
26．王振文，董霞．1985．仲丁胺及其衍生物对于多种水果、蔬菜贮藏期防腐保鲜效果的研究．河北农业大学学报．8(3)：23-35
27．吴潇．2001．薄层色谱法在氯霉素片含量分析中的应用．药物分析杂志．21(5)：368
28．熊小敏．1 997．薄层-溴化法测定福美双和福美锌含量．农资科技．(5)：23-24
29．杨文熙，陈辉平，张瑞敏，范志斌．1985．低级脂肪胺的气相色谱分析．河北农业大学学报．8(3)：72-81
30．叶凤阁，张忠源．1991．仲丁胺抑菌作用及其机理．北方园艺．(2)：15-18
31．俞加林，赵敏，黄湘倚．1991．国内水果保鲜剂研究进展．北方园艺．69(1)：35-37
32．张胜强，许清．1995．TLC-UV法测定小鼠体内的安定．中国药科大学学报．26(1)：20-22
33．张晓平，杨国林，何庆，米沙．2002．柱前衍生化反相高效液相色谱法测定N-甘氨酰
-L-谷氨酰胺—水合物含量．药物分析杂志．22(2)：89-91
34．张忆华，杨家顺．2001．食品中的农药残留及一些对策．食品发酵工业．27(7)：68-71
35．张志云，田勇，宋壮兴，冯晓元．1996．几种防腐剂对梨贮藏保鲜效果的实验．北方果树．(3)：13-15
36．甄增立，郑树林．1985．仲丁胺处理的蒜苔经贮运后残留量分析．河北农业大学学报．8(3)：130-136
37．周同惠．1989．纸色谱和薄层色谱．北京：科学出版社
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