CN111855847A - 高效液相色谱法测定富硒蛋白多糖中总硒含量的方法 - Google Patents
高效液相色谱法测定富硒蛋白多糖中总硒含量的方法 Download PDFInfo
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Abstract
本发明公开了一种高效液相色谱法测定富硒蛋白多糖中总硒含量的方法。该方法以2,3—二氨基萘为螯合剂,以400µL乙腈为分散剂,120µL氯苯为萃取剂,硒(IV)与2,3—二氨基萘的螯合物(4,5‑苯并苤硒脑)经乙腈稀释后直接采用PFP色谱柱分离,乙腈洗脱,用荧光检测器在激发波长为376 nm、发射波长为520 nm条件下测定荧光强度,外标法定量。本方法具有实验结果稳定、样品转移少、试剂用量少、回收率高等特点。
Description
技术领域
本发明涉及一种高效液相色谱法测定富硒蛋白多糖中总硒含量的方法。
背景技术
硒(Se)作为动物生命必需微量元素和较窄的安全阈值以及其不同生物活性依赖于不同的存在形态等原因,对硒在不同基质中的分析方法研究一直引起广泛的关注。此外,亚硒酸盐和有机硒化合物作为一种饲料添加剂加入饲料中。因此,建立可靠的硒测定方法是非常有必要的。目前测定硒含量有各种高选择性的分析技术,包括原子吸收光谱法(AAS)、原子发射光谱法(AES)、电感耦合等离子体质谱法(ICP-MS)]和大气压化学电离质谱法(APCI-MS)等,这些技术常用于测定土壤、食品、水和生物样品中的硒含量。然而,这些技术需要昂贵的设备,普通实验室很难配置。最常规的硒测定方法是荧光测定技术,使用2,3—二氨基萘(DAN)作为衍生化试剂与硒(IV)生成硒螯合物(4,5-苯并苤硒脑,Se-DAN),再利用荧光分光光度计检测。高效液相色谱法用于许多领域的常规分析,其成本为相对较低,衍生法用以提高仪器检测灵敏度。除此之外,因样品中基质干扰,使得硒含量难以得到准确测定。因此,往往需要对样品有一个提取和浓缩的前处理步骤。迄今为止,对于硒螯合物的前处理方法主要有液液萃取法和分散液液微萃取法,液液萃取法主要是以环己烷作为萃取剂,在硒(IV)与2, 3—二氨基萘生成螯合物后,对4,5-苯并苤硒脑进行萃取,而分散液液微萃取法是以乙腈为分散剂,氯苯为萃取剂,让体系形成萃取液滴,实现提取和预浓缩一步到位。Zhou等利用液液分散微萃取和反相C18柱高效液相色谱紫外检测法测定了茶叶中总硒含量,但是在应用此方法对硒蛋白多糖中总硒含量测定过程中发现,前处理过程中对萃取剂的浓缩会造成4,5-苯并苤硒脑的损失,更重要的一点是普通C18对于4,5-苯并苤硒脑和体系中的干扰物并不能有效分离。
发明内容
为了克服现有技术中的问题,本发明提供了高效液相色谱法测定富硒蛋白多糖中总硒含量的方法,快速、环保、简便、精准。
一种高效液相色谱法测定富硒蛋白多糖中总硒含量的方法,富硒蛋白多糖采用HNO3—HClO4体系消解后,以2, 3—二氨基萘为螯合剂,乙腈为分散剂,氯苯为萃取剂,硒(IV)与2, 3—二氨基萘的螯合物Se-DAN经乙腈1:1稀释后采用PFP色谱柱分离,乙腈洗脱,用荧光检测器在激发波长为 376 nm、发射波长为 520 nm 条件下测定荧光强度,外标法定量。
所述的方法,称取25 mg 富硒蛋白多糖样品,加 10 mL HNO3-HClO4 混合酸,其中内含9ml HNO3和1ml HClO4,冷消化过夜;12 h 后于电热板上加热消解至剩余体积 2 mL ,冷却后再加入 5 mL 6 mol/L盐酸溶液,继续加热至剩余体积 2 mL,冷却后调节pH值1.5~2.0之间,并用水定容至 250 mL,取出3 mL样品至5 mL具塞离心管中,向其中加入0.5 mLDAN,沸水浴反应10分钟,流水冷却后,再向其中先后加入400 μL乙腈和120 μL氯苯,振摇5分钟,然后静置5分钟,最后以5000 rpm/min的转速,离心5分钟;移取100 μL氯苯并向其中加入100 μL乙腈,混匀后进样,经PFP色谱柱分离,串联紫外与荧光检测器(RPHPLC-UV-FLD)检测分析。
本发明的有益效果:
发明利用微量的分散剂和萃取剂对液液分散微萃取实现对样品中的Se-DAN进行了萃取,接下去对于萃取剂不需要常规浓缩,直接稀释后进样分析,防止氮吹浓缩过程中产生的荧光猝灭;采用PFP色谱柱在乙腈为流动相条件下,有效避免了富硒蛋白多糖样品中基质的干扰,实现了对富硒蛋白多糖样品中Se-DAN准确定量并且避免了样品多次转移,有机溶剂的大量使用。
附图说明
图1为样品溶液色谱图。
图2为亚硒酸钠标准溶液的标准曲线及线性方程。
图3 PFP色谱柱(上)和C18色谱柱(下)分析富硒蛋白多糖样品中Se-DAN螯合物色谱图;
分析条件:流速0.3 mL/min,紫外吸收波长378 nm。
图4 PFP色谱柱(上)和C18色谱柱(下)分析富硒蛋白多糖样品中Se-DAN螯合物色谱图;
分析条件:流速0.3 mL/min,紫外吸收波长378 nm,激发波长376 nm,发射波长520 nm。
具体实施方式
以下结合附图和实施例对本发明进一步详细的阐述。
方法原理
利用待测物在样品溶液和萃取剂中的溶解度不同从而实现分离富集。在实验操作过程中,首先向离心管中的待测溶液中加入混合均匀的萃取剂和分散剂,使整个体系形成浑浊体系,再通过离心机进行离心分层后通过微量进样器取出下层有机相进行检测分析。
取出消解后的样品3 mL,加至5 mL具塞离心管中,向其中加入0.5 mL DAN,沸水浴反应10分钟,流水冷却后,再向其中先后加入400 μL乙腈和120 μL氯苯,振摇5分钟,然后静置5分钟,最后以5000 rpm/min的转速,离心5分钟。移取100 μL氯苯并向其中加入100 μL乙腈,混匀后进样,经RPHPLC-UV-FLD分析。
仪器与试剂
数显恒温水浴锅(常州国华电器有限公司),pH计(美国Mettler Toledo公司),Softmaxpro5多功能连续光谱酶标仪(美国Molecular Devices公司),纯水仪(日本Yamato公司),Ultimate 3000高效液相色谱仪配置紫外检测器和荧光检测器(美国Thermo Scientific公司)、高速离心机(美国Thermo Scientific公司)。亚硒酸钠标准品(Na2SeO3,纯度99%)、乙腈(色谱纯)、2,3-二氨基萘(DAN,纯度95%)从sigma公司购买。EDTA二钠、盐酸羟胺、盐酸、硝酸、高氯酸、氯苯等试剂均为分析纯,购自国药集团化学试剂有限公司。
DAN 试剂(1.0 g/L)的配制:参照GB 13883-2008《饲料中硒的测定》。
分析条件
Welch Ultimate PFP色谱柱(250 mm×4.6 mm,3 μm,中国月旭公司);Xbrige色谱柱(250 mm×4.6 mm,5 μm,美国waters公司);进样量5 μL;流动相:乙腈。UV检测器,波长378nm;FLD检测器,激发波长376 nm,发射波长520 nm。
富硒蛋白多糖由本实验室发酵(Xu,2009,略有修改),对于本领域技术人员来说可以自制或者外购合适的富硒蛋白多糖样品。
实施例1 标准曲线的绘制
准确称取量50.0 mg亚硒酸钠溶解在500 mL 0.1 mol/L HCl中,用0.1 mol/L HCl逐步稀释成500.0、250.0、100.0、50.0和25.0 µg/L的亚硒酸钠标准工作液。经过0005步骤操作后,利用RPHPLC-UV-FLD分析测定4,5-苯并苤硒脑含量。
实施例2 国标法测定总硒含量
准确称取 0.5 g~3 g 样品,加 10 mL HNO3-HClO4 混合酸(9+1)冷消化过夜。12 h 后于电热板上加热消解至剩余体积 2 mL 左右,冷却后再加 5 mL 盐酸溶液(6 mol/L),继续加热至剩余体积 2 mL 左右。冷却后用水定容至 100 mL,量取部分溶液(pH 1.5~2.0,Se含量≤0.4 µg)加入3 mL 盐酸羟胺和2 mL DAN,摇匀,沸水浴5 min。冷却后用10 mL 环己烷萃取,用荧光光度计在激发波长为 376 nm、发射波长为 520 nm 条件下测定荧光强度,从而计算出试样中的硒含量。
实施例3富硒蛋白多糖中总硒含量测定
称取25 mg 富硒蛋白多糖样品,加 10 mL HNO3-HClO4 混合酸(9ml HNO3和1ml HClO4)冷消化过夜;12 h 后于电热板上加热消解至剩余体积 2 mL ,冷却后再加入 5 mL 盐酸溶液(6 mol/L),继续加热至剩余体积 2 mL ,冷却后调节pH值1.5~2.0之间,并用水定容至250 mL,取出3 mL样品至5 mL具塞离心管中,向其中加入0.5 mL DAN,沸水浴反应10分钟,流水冷却后,再向其中先后加入400 μL乙腈和120 μL氯苯,振摇5分钟,然后静置5分钟,最后以5000 rpm/min的转速,离心5分钟;移取100 μL氯苯并向其中加入100 μL乙腈,混匀后进样,经PFP色谱柱分离,串联紫外与荧光检测器(RPHPLC-UV-FLD)检测分析。
根据以上实施例1-3得到的结果:
RPHPLC-FLD色谱图
PFP色谱柱在流速为0.3 mL/min的条件下分析Na2SeO3标准品与2, 3—二氨基萘螯合物(Se-DAN)色谱图如图1所示。
标准曲线的绘制
以各浓度为横坐标(x),荧光检测积分面积为纵坐标(y),拟合计算得到Na2SeO3的标准曲线方程及相关系数,如图2所示。线性方程式为:y=4921.08x-9803.09,相关系数为R2=0.9994。
比较PFP色谱柱与C18色谱柱以及不同检测器分析Se-DAN
如图3所示实验结果表明:无论使用PFP色谱柱还是C18色谱柱,用紫外检测器都不能对富硒蛋白多糖样品中Se-DAN进行准确定量,估计是样品中的具有紫外吸收的物质造成的基质干扰。而如图3所示,选择PFP色谱柱,用荧光检测器时,样品中的Se-DAN能呈现出很好的峰形,这一点在C18色谱柱上则不能实现,所以最终选择PFP色谱柱和荧光检测器。
氮气吹干浓缩对结果的影响
Zhou等对萃取液进行氮吹浓缩至干再用甲醇复溶后经HPLC分析,本发明在对萃取液氮气吹干浓缩过程中发现,浓缩过程尤其是吹干结束时,空气对Se-DAN荧光的猝灭影响很大。因此,为了避免氮吹浓缩过程空气对荧光的猝灭,同时,萃取相还与流动相体系互溶,本发明直接采用乙腈对萃取剂进行1:1稀释后进样,表现出良好的线性(如图2所示)和准确度(如表1所示)。并且如表2利用RPHPLC-FLD 10小时前后测定Se-DAN结果所示,发现Se-DAN螯合物在室温下放置10小时,仍然十分稳定。
国标法测定总硒含量结果
采用国标法GB/T 13883-2008 (饲料中硒的测定)测定富硒蛋白多糖中硒的含量为1327.8 µg/g,与本方法测定结果高度一致。
富硒蛋白多糖中总硒含量测定结果
5次测得富硒蛋白多糖中硒含量平均值为1334.9 µg/g,相对标准偏差为3.50%,如表1所示。
Claims (2)
1.一种高效液相色谱法测定富硒蛋白多糖中总硒含量的方法,其特征是:富硒蛋白多糖采用HNO3—HClO4体系消解后,以2, 3—二氨基萘为螯合剂,乙腈为分散剂,氯苯为萃取剂,硒(IV)与2, 3—二氨基萘的螯合物Se-DAN经乙腈1:1稀释后采用PFP色谱柱分离,乙腈洗脱,用荧光检测器在激发波长为 376 nm、发射波长为 520 nm 条件下测定荧光强度,外标法定量。
2.根据权利要求1所述的方法,其特征是:称取25 mg 富硒蛋白多糖样品,加 10 mLHNO3-HClO4 混合酸,其中内含9ml HNO3和1ml HClO4,冷消化过夜;12 h 后于电热板上加热消解至剩余体积 2 mL ,冷却后再加入 5 mL 6 mol/L盐酸溶液,继续加热至剩余体积 2mL,冷却后调节pH值1.5~2.0之间,并用水定容至 250 mL,取出3 mL样品至5 mL具塞离心管中,向其中加入0.5 mL DAN,沸水浴反应10分钟,流水冷却后,再向其中先后加入400 μL乙腈和120 μL氯苯,振摇5分钟,然后静置5分钟,最后以5000 rpm/min的转速,离心5分钟;移取100 μL氯苯并向其中加入100 μL乙腈,混匀后进样,经PFP色谱柱分离,串联紫外与荧光检测器(RPHPLC-UV-FLD)检测分析。
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