离子色谱柱切换在线前处理法同时测定皮革及织物中的三价铬与六价铬离子

贺婕等
摘要采用一根NG1反相色谱柱作为前处理柱在线去除样品中的水溶性有机基质, 建立了离子色谱柱切换技术同时测定Cr与Cr的方法。进样前,先将待测样品水溶液与一定浓度的乙二胺四乙酸(EDTA)溶液充分反应,使其中的Cr络合生成阴离子产物,该阴离子产物在可见光范围内有较强吸收;进样后,样品中的离子经前处理柱分离后被收集在2 mL接收环内,通过柱切换技术,淋洗液将接收环内的离子带至阴离子分析柱中分离,Cr与1,5二苯卡巴肼(DPC)溶液进行衍生化反应后与Cr的EDTA络合物在同一波长下有较强吸收,由此可完成对两种离子的同波长测定。
1引言
铬元素广泛存在于自然界中,常以Cr和Cr两种形式存在。其中Cr是人体必需的微量元素,在正常食品补给剂量下无毒,但较高浓度则表现出细胞毒性反应;Cr易穿过细胞膜被人体吸收,干扰人体内的正常生理过程,是致癌和致突变的诱发因子,浓度大时会导致死亡\[1\]。正因二者具有不同的毒副作用,建立针对不同价态铬离子的测定方法就显得尤为重要。
目前针对样品中不同价态的铬,有氧化还原法,分光光度法,原子吸收光谱法等检测方法。比较几种方法,利用过差减法进行的氧化还原法原理简单且成本低,但反应时间长,需严格控制溶液酸度,防止Cr与Cr相互转变,易使检测结果出现误差;分光光度法与原子吸收光谱法发展较快,应用广泛,其中分光光度法在测定Cr时存在基质干扰,导致结果不准确\[2~4\],而原子吸收光谱本身无法分辨Cr与Cr,需借助硅胶等介质,吸附后分别洗脱检测,操作复杂且成本较高\[5,6\]。近几年,利用色谱高效的分离性能与几种检测器相结合来测定不同价态铬的方法有了较大的进展[7],其中离子交换色谱\[8~11\]和反相离子对色谱\[12,13\]应用较多。
皮革是一种日常生活中经常接触的物质,因其鞣制过程用到铬,而被列入了铬含量测定的黑名单。国际上对皮革中Cr的含量有严格的控制,通用的测定标准一般是先将粉碎的皮革浸提,后加入DPC还原生成紫红色配合物,用紫外可见分光光度法进行测定。但浸提液中存在的水溶性有机物如染料,严重干扰了目标离子的测定。对此,国内外科学家已进行大量研究,解决方法主要有毛细管电泳法预分离\[14\]、固相萃取柱除有机基质\[15,16\]以及加入有机脱色剂处理\[17\],但这些方法均因成本高、操作复杂或检测灵敏度低等缺点,没有得到广泛应用。本方法利用柱切换技术,于分离系统之前增加了样品前处理系统,除去样品中存在的水溶性有机基质,实现了对样品的在线前处理,省去繁琐的样品前处理过程,减少了人力消耗。与已有方法相比,本方法增加了对Cr的柱前衍生,将待测样品水溶液与EDTA溶液充分反应,使Cr络合生成在可见光区有强吸收的阴离子基团,与未络合的Cr一并进入阴离子色谱柱中分离,Cr在柱后衍生生成在可见光区同样有较强吸收的基团,由此完成对Cr与Cr的同时检测,应用范围更广,对于样品中铬的测定也更加完善。
2实验部分
2.1仪器与试剂
SHCIC离子色谱仪(中国青岛盛瀚色谱技术有限公司),配有一个六通进样阀及100 μL定量环,两个UC3281 HPLC泵,一个柱后衍生装置;实验室自制阴离子柱\[18\],Dionex IonPac NG1(35 mm ×4 mm)反相柱(美国赛默飞世尔科技有限公司);ZQH100柱切换器(中国青岛盛瀚色谱技术有限公司);UV3292紫外可见检测器(中国北京优联光电技术有限公司);P680高效液相色谱四源梯度泵(美国赛默飞世尔科技有限公司);JY92Ⅱ超声细胞粉碎仪(中国宁波新芝生物科技股份有限公司);UV2550型紫外可见分光光度计(日本岛津仪器公司);PHSJ5型pH酸度计(上海仪电科学仪器股份有限公司); 0.22 μm尼龙滤膜过滤头。
重铬酸钾(K2Cr2O7,分析纯,上海浦江化工厂);氯化铬(CrCl3·6H2O,分析纯,天津市东丽区天大化学试剂厂);乙二胺四乙酸二钠(EDTA·2Na,分析纯,北京化工厂);1,5二苯卡巴肼(DPC,分析纯,国药集团化学试剂有限公司);50% (w/w) NaOH(美国赛默飞世尔科技有限公司); Na2HPO4·12H2O(分析纯,国药集团化学试剂有限公司);浓H2SO4(衢州巨化试剂有限公司);乙腈、甲醇(色谱纯,德国默克公司);实验室用水为18.2 MΩ·cm去离子水(美国密理博公司)。
2.2标准溶液配制及样品前处理
1000 mg/L Cr标准溶液:准确称取0.51 g CrCl3·6H2O于100 mL容量瓶中,以水溶解并定容。
1000 mg/L Cr标准溶液:准确称取0.28 g K2Cr2O7于100 mL容量瓶中,以水溶解并定容,即得。
称取0.37 g EDTA溶于10 mL水中, 得100 mmol/L EDTA溶液。称取质量分数为50% NaOH 0.4 g, 用去离子水稀释至10 mL, 得0.5 mmol/L NaOH溶液。称取0.4 g DPC于100 mL甲醇中,边搅拌边缓慢加入8 mL浓H2SO4,用水定容至1 L, 即得柱后衍生液。
20 mmol/L NaOH溶液:称取质量分数为50 %的NaOH 1.6 g,用水定容至1 L,该淋洗液在氮气保护下现配现用。
磷酸盐缓冲溶液(PBS):称取约3.58 g Na2HPO4于100 mL容量瓶中,用水溶解并定容,用H3PO4调节缓冲溶液至pH=8.0±0.1。
Cr与Cr混合标样:取不同体积Cr与Cr标准溶液,用磷酸盐缓冲溶液稀释至5 mL,加入0.5 mL 100 mmol/L EDTA溶液与67 μL 0.5 mol/L NaOH溶液,用水定容至10 mL,反应1 h,经0.22 μm尼龙滤膜过滤,收集滤液待用。
样品前处理:称取剪碎的样品约0.30 g,加入10 mL磷酸盐缓冲溶液,在超声细胞粉碎仪中超声萃取约3 h,取5 mL溶液,加入1 mL 100 mmol/L EDTA溶液与0.2 mL 0.5 mol/L NaOH溶液,用水定容至10 mL,反应约1 h,经0.22 μm尼龙滤膜过滤,收集滤液待用。
2.3实验装置
整个检测过程可分为4个步骤:(1)进样:注射进样,使阀1上固定的100 μL定量环充满待测样品;(2)0.0~2.5 min样品中有机基质的在线去除:切换阀1至Inject,当待测样品随着泵1泵入的NaOH淋洗液经过NG1前处理柱时,样品中的有机物基质被保留在柱中,无保留作用的离子则随着淋洗液被固定于阀2上的2 mL接收管收集;(3)2.5~20.0 min目标离子的分析和前处理柱的再生:切换阀1至Load,阀2至Inject,接收管内的待测离子由泵2泵入的NaOH淋洗液带至阴离子分析柱中分离,同时,前处理柱以乙腈为淋洗液,洗去柱中保留的有机物基质,进行再生;(4)20~25.0min平衡系统:前处理柱换NaOH为淋洗液,使前处理柱在下一次进样前得到平衡。最后,Cr的EDTA络合物与经DPC衍生的Cr在545 nm波长下进行吸光度检测,阀2切回Load。色谱仪器装置图如图1所示,阀上实线所示为Load状态,而虚线为Inject状态。
3结果与讨论
3.1样品萃取条件选择
分别以水、pH=8的磷酸盐缓冲溶液、20 mmol/L NaOH溶液为萃取液,提取样品中的Cr与Cr,前处理方法同2.2节。比较3种溶液的萃取效果并参考文献\[19\]可知,当pH值过高时,Cr易生成沉淀析出,故以20 mmol/L NaOH为萃取液时,Cr的检出浓度相对最小。同时参考国标方法\[20\],皮革及织物因与皮肤接触而产生危害,宜采用模拟人体汗液体系为萃取液较为合适,因此在本实验中将磷酸盐缓冲溶液(pH=8.0±0.1)选为萃取液。
3.2分析检测条件选择
本实验利用柱切换技术实现对样品萃取液中有机物基质在线去除。为保证前处理柱中洗脱的离子均能被接收管收集,设计以下实验:在未接阴离子分析柱的条件下,经前处理柱洗脱的离子与衍生液反应后直接进入紫外可见检测器检测,离子出峰保留时间在0.9~2.0 min,淋洗液流速为1 mL/min,为避免样品离子在接收管中扩散引起损失,阀2的切换时间为2.5 min,接收管体积为2 mL。
对Cr标样与EDTA衍生产物及Cr标样与DPC衍生产物进行紫外可见全波长扫描,确定二者同时检测波长为545 nm。
优化Cr与EDTA溶液衍生反应条件,分别考察NaOH浓度、EDTA浓度, 以及反应温度和反应时间4个影响因素。
利用分光光度法测定Cr与DPC衍生产物吸光度值,完成对Cr的定量方法已比较成熟\[2\] 。本实验考察DPC浓度及酸度对反应影响,如图3所示。保持H2SO4与甲醇浓度不变,配制一系列不同浓度的DPC衍生液,确定浓度为0.4 g/L时,响应较大且噪音小;考察硫酸浓度对衍生反应的影响,在H2SO4浓度较低时,衍生反应慢,无法达到在线衍生检测的要求,而在H2SO4浓度升至0.15 mol/L时,生成衍生物速度较快且信号值相对较为稳定,重现性好。
2Abdollahi H. Anal. Chim. Acta, 2001, 442 (2): 327-336
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9Chen Z L, Megharaj M, Naidu R. Talanta, 2007, 73 (5): 948-952
10ZHU Min, LIN ShaoMei, YAO Qi, XU ZiGang, ZHU Yan. Journal of Zhejiang University (Science Edition), 2007, 34(3): 326-329
朱 敏, 林少美, 姚 琪, 徐子刚, 朱 岩. 浙江大学学报(理学版), 2007, 34(3): 326-329
11Yang Y, He J, Huang Z, Zhong N, Zhu Z, Jiang R, You J, Lu X, Zhu Y, He S. J. Chromatogr. A, 2013, 1305 (1): 171-175
12Wolf R E, Morrison J M, Goldhaber M B. J. Anal. At. Spectrom., 2007, 22 (9): 1051-1060
13Kuo C Y, Jiang S J, Sahayam A C. J. Anal. At. Spectrom., 2007, 22 (6): 636-641
14WANG BaiSong, WAN JunSheng, DING FuXin, WANG WuShang. Journal of Instrumental Analysis, 2006, 25(6): 56-59
王柏松, 万俊生, 丁富新, 王武尚. 分析测试学报, 2006, 25(6): 56-59
15NIU ZengYuan, YE XiWen, WANG YingJie, WANG Yan, JIAO LiZhen, WANG JingTang. China Leather, 2006, 35(11): 35-38
牛增元, 叶曦雯, 王英杰, 王 岩, 矫丽珍, 王境堂. 中国皮革, 2006, 35(11): 35-38
16TONG ChengBao. Westleather, 2013, 35(12): 40-42
童成豹. 西部皮革, 2013, 35(12): 40-42
17SHEN Bing, GAN DeFen, YU XuFeng. China Leather, 2003, 32(21): 29-33
沈 兵, 干德芬, 俞旭峰. 中国皮革, 2003, 32(21): 29-33
18Huang Z, Zhu Z, Subhani Q, Yan W, Guo W, Zhu Y. J. Chromatogr. A, 2012, 1251: 154-159
19Woittiez R W, Lagerwaard A, Volkers K J, Tangonan M D C. J. Radioanal. Nucl. Chem., 1993, 169(1): 229-237
20GB/T 228072008, Leather and furChemical testsDetermination of chromium Ⅵ content, National Standards of the People′s Republic of China
皮革和毛皮化学实验六价铬含量的测定, 中华人民共和国国家标准, GB/T 228072008
AbstractA new analytical method has been developed for the simultaneous determination of Cr and Cr using online sample pretreatment valveswitching ion chromatography. The organic matrix in leather was removed by using a reversephase column as the pretreatment column. Before injection, EDTA was added into sample solution to react with the Cr to form anion which could absorb visible light strongly. After injection, the ions separated by the pretreatment column were received in a collection loop. Then the ions were delivered into an analytical column and separated. Cr then was derived with the derivatization reagent 1,5diphenylcarbazide (DPC), and detected together with CrEDTA complex by a UVVis detector. Under the optimum conditions, the linear range of the method for Cr and Cr was 0.3-10 mg/L (r=0.9991) and 0.05-2 mg/L(r=0.9992), whereas detection limits (S/N=3) were 80.78 μg/L and 6.67 μg/L, respectively. The recoveries were in the range of 88.7%-108.5% with the relative standard deviations for retention time and peak area less than 3%. The method could be applied to determine Cr and Cr in leather and cloth effectively and quickly.
KeywordsIon chromatography; Column switching; Online pretreatment; Chromium
11Yang Y, He J, Huang Z, Zhong N, Zhu Z, Jiang R, You J, Lu X, Zhu Y, He S. J. Chromatogr. A, 2013, 1305 (1): 171-175
12Wolf R E, Morrison J M, Goldhaber M B. J. Anal. At. Spectrom., 2007, 22 (9): 1051-1060
13Kuo C Y, Jiang S J, Sahayam A C. J. Anal. At. Spectrom., 2007, 22 (6): 636-641
14WANG BaiSong, WAN JunSheng, DING FuXin, WANG WuShang. Journal of Instrumental Analysis, 2006, 25(6): 56-59
王柏松, 万俊生, 丁富新, 王武尚. 分析测试学报, 2006, 25(6): 56-59
15NIU ZengYuan, YE XiWen, WANG YingJie, WANG Yan, JIAO LiZhen, WANG JingTang. China Leather, 2006, 35(11): 35-38
牛增元, 叶曦雯, 王英杰, 王 岩, 矫丽珍, 王境堂. 中国皮革, 2006, 35(11): 35-38
16TONG ChengBao. Westleather, 2013, 35(12): 40-42
童成豹. 西部皮革, 2013, 35(12): 40-42
17SHEN Bing, GAN DeFen, YU XuFeng. China Leather, 2003, 32(21): 29-33
沈 兵, 干德芬, 俞旭峰. 中国皮革, 2003, 32(21): 29-33
18Huang Z, Zhu Z, Subhani Q, Yan W, Guo W, Zhu Y. J. Chromatogr. A, 2012, 1251: 154-159
19Woittiez R W, Lagerwaard A, Volkers K J, Tangonan M D C. J. Radioanal. Nucl. Chem., 1993, 169(1): 229-237
20GB/T 228072008, Leather and furChemical testsDetermination of chromium Ⅵ content, National Standards of the People′s Republic of China
皮革和毛皮化学实验六价铬含量的测定, 中华人民共和国国家标准, GB/T 228072008
AbstractA new analytical method has been developed for the simultaneous determination of Cr and Cr using online sample pretreatment valveswitching ion chromatography. The organic matrix in leather was removed by using a reversephase column as the pretreatment column. Before injection, EDTA was added into sample solution to react with the Cr to form anion which could absorb visible light strongly. After injection, the ions separated by the pretreatment column were received in a collection loop. Then the ions were delivered into an analytical column and separated. Cr then was derived with the derivatization reagent 1,5diphenylcarbazide (DPC), and detected together with CrEDTA complex by a UVVis detector. Under the optimum conditions, the linear range of the method for Cr and Cr was 0.3-10 mg/L (r=0.9991) and 0.05-2 mg/L(r=0.9992), whereas detection limits (S/N=3) were 80.78 μg/L and 6.67 μg/L, respectively. The recoveries were in the range of 88.7%-108.5% with the relative standard deviations for retention time and peak area less than 3%. The method could be applied to determine Cr and Cr in leather and cloth effectively and quickly.
KeywordsIon chromatography; Column switching; Online pretreatment; Chromium
11Yang Y, He J, Huang Z, Zhong N, Zhu Z, Jiang R, You J, Lu X, Zhu Y, He S. J. Chromatogr. A, 2013, 1305 (1): 171-175
12Wolf R E, Morrison J M, Goldhaber M B. J. Anal. At. Spectrom., 2007, 22 (9): 1051-1060
13Kuo C Y, Jiang S J, Sahayam A C. J. Anal. At. Spectrom., 2007, 22 (6): 636-641
14WANG BaiSong, WAN JunSheng, DING FuXin, WANG WuShang. Journal of Instrumental Analysis, 2006, 25(6): 56-59
王柏松, 万俊生, 丁富新, 王武尚. 分析测试学报, 2006, 25(6): 56-59
15NIU ZengYuan, YE XiWen, WANG YingJie, WANG Yan, JIAO LiZhen, WANG JingTang. China Leather, 2006, 35(11): 35-38
牛增元, 叶曦雯, 王英杰, 王 岩, 矫丽珍, 王境堂. 中国皮革, 2006, 35(11): 35-38
16TONG ChengBao. Westleather, 2013, 35(12): 40-42
童成豹. 西部皮革, 2013, 35(12): 40-42
17SHEN Bing, GAN DeFen, YU XuFeng. China Leather, 2003, 32(21): 29-33
沈 兵, 干德芬, 俞旭峰. 中国皮革, 2003, 32(21): 29-33
18Huang Z, Zhu Z, Subhani Q, Yan W, Guo W, Zhu Y. J. Chromatogr. A, 2012, 1251: 154-159
19Woittiez R W, Lagerwaard A, Volkers K J, Tangonan M D C. J. Radioanal. Nucl. Chem., 1993, 169(1): 229-237
20GB/T 228072008, Leather and furChemical testsDetermination of chromium Ⅵ content, National Standards of the People′s Republic of China
皮革和毛皮化学实验六价铬含量的测定, 中华人民共和国国家标准, GB/T 228072008
AbstractA new analytical method has been developed for the simultaneous determination of Cr and Cr using online sample pretreatment valveswitching ion chromatography. The organic matrix in leather was removed by using a reversephase column as the pretreatment column. Before injection, EDTA was added into sample solution to react with the Cr to form anion which could absorb visible light strongly. After injection, the ions separated by the pretreatment column were received in a collection loop. Then the ions were delivered into an analytical column and separated. Cr then was derived with the derivatization reagent 1,5diphenylcarbazide (DPC), and detected together with CrEDTA complex by a UVVis detector. Under the optimum conditions, the linear range of the method for Cr and Cr was 0.3-10 mg/L (r=0.9991) and 0.05-2 mg/L(r=0.9992), whereas detection limits (S/N=3) were 80.78 μg/L and 6.67 μg/L, respectively. The recoveries were in the range of 88.7%-108.5% with the relative standard deviations for retention time and peak area less than 3%. The method could be applied to determine Cr and Cr in leather and cloth effectively and quickly.
KeywordsIon chromatography; Column switching; Online pretreatment; Chromium
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