修饰电极检测植物油过氧化值的研究
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摘要
本课题以植物油过氧化值为检测指标,采用生物及化学修饰法分别制备两类电极,研究它们在有机相介质中的电化学行为,探讨反应机理及电极反应过程,确定最佳的技术参数,以构建良好的催化反应微环境及获得较高的电子传递速率,使其能够满足植物油检测的实际需要。同时将研究确定的方法应用到微小电极上,开发能够快速、准确定性、定量的植物油过氧化值检测技术及设备。主要研究内容如下：
1.采用溶胶-凝胶法构建的双层膜结构HRP酶电极,在有机相介质中能够对过氧化月桂酰产生显著地还原电流响应,作为电子介体的亚铁氰化钾能够有效的在HRP酶与电极表面传递电子,增强还原电流响应；随着过氧化月桂酰添加量的增加,HRP酶电极产生的还原电流逐渐增强,氧化电流逐渐减弱：通过对还原峰电流与扫描速度间的关系研究,证明当扫描速度较低时HRP酶电极以传质过程控制为主,即通过HRP酶传递到电极表面发生还原反应产生还原电流。当扫描速度较高时,电极的传质过程控制转弱,逐渐转为表面过程控制,此时HRP酶电极的氧化还原反应与电子介体——亚铁氰化钾在电极表面的氧化还原有关；采用计时电流法根据不同浓度的过氧化月桂酰在HRP酶电极上的催化还原电流,建立其与过氧化物浓度间的校正曲线,并根据Lineweaver-Burk方程,确定该体系下HRP酶的表观米氏常数Km。
2.考察了碘离子与壳聚糖在石墨电极表面的电极反应过程属吸附溶出伏安过程,即碘离子与质子化的壳聚糖结合,生成的新物质在电极表面得到富集,通过由负向正的电位扫描,电极表面的碘离子失去电子,被氧化成游离的碘单质,无法被壳聚糖吸附,进而从电极表面脱附出来,同时产生氧化峰电流；最佳的碘离子测定条件为：以0.1mol/L柠檬酸-柠檬酸钠缓冲液(pH5.0)为底液,壳聚糖浓度200μg/mL;吸附溶出参数：富集电位OV,富集时间180s,而静止时间、静止电位对峰电流的影响较小,这里采用的是静止电位OV,静止时间25s；微分脉冲伏安法扫描参数：开始电位0.1V,终止电位1.5V,电位增量0.004V,振幅0.05V,脉冲宽度0.1s,脉冲周期0.2s；在此条件下,石墨电极产生的氧化峰电流与碘离子浓度在0.07-0.49g/L内,呈良好的线性相关关系(R=0.9990)。该方法具有一定的抗干扰能力,表现出良好的重现性及稳定性。使用后的电极经过一定的再生活化步骤即可重复使用。
3.阐述了壳聚糖修饰丝网印刷电极催化反应机理,确定了采用微分脉冲溶出伏安法测定植物油过氧化值的条件。由于质子化的壳聚糖只能吸附碘离子,因此壳聚糖修饰电极可以用来确定碘离子的氧化过程。结果表明,峰电流值随植物油过氧化值的升高而降低,且在0.01-3.21μg/mL范围内呈良好的相关关系(R=0.9914)。此外,将采用壳聚糖修饰电极测定得到的过氧化值与标准碘量法的测定结果进行初步比对,结果基本一致。
4.课题组研究设计一种检测过氧化值的新方法,即采用壳聚糖修饰丝网印刷电极经微分脉冲溶出伏安法扫描,来取代碘量法中人工滴定确定碘离子浓度变化的步骤。本研究采用上述方法对40种市售植物油的过氧化值进行测定,并与国标法(GB/T5538-2005)的测定结果进行比对,综合偏离度与En数两项指标进行评价,结果表明：两种方法在定性判定方面结果一致,而在定量判断方面有33个结果满意,整体满意率为82.5%,其中0.031～0.090meq/kg及0.096～0.150meq/kg区间内的结果更为准确,满意率分别为82.35%及93.33%。研究表明,与国标法相比,新方法在操作性、安全性等方面存在一定优势,在一定过氧化值范围内,结果准确性得到认可。
This research choosed peroxide value of vegetable oil as detection index, used the biologic al and chemical modification method to make two kinds of electrodes respectively, studied their electrochemical behavior in organic phase medium, discussed the reaction mechanism and the electrod e reaction process, and determined the best technical parameters. By these parameters, a good catalytic r eaction micro environment was built to meet the practical requirement of vegetable oil testing. In the me antime. this method was applied to the micro-electrodes (screen-printed electrodes), and detection tech nology and equipment which can quantify the vegetable oil quickly and accurately were developed. It s main contents are as follows:
1. The HRP enzyme electrode with a double-film structure constructed by using the Sol-Gel method can generate a significant reduction current response to lauroyl peroxide in an organic phase medium, and potassium ferrocyanide as the electron mediator can effectively transfer electrons between the HRP enzyme and the electrode surface, and enhance reduction current responses. As the added amount of lauroyl peroxide increased, the reduction current generated by the HRP enzyme electrode gradually increased, and the oxidation current gradually weakened; Through exploration of the relationship between the reduction peak current and the scanning speed, it is demonstrated that when the scanning speed is low, the HRP enzyme electrode mainly involves the mass transfer process control, that is, the reduction reaction is carried out by transfer of the HRP enzyme onto the electrode surface to generate reduction current. When the scanning speed is high, the mass transfer process control of the electrode weakens and is gradually turned into the surface process control, in which the oxidation-reduction reaction of the HRP enzyme electrode is relevant to the oxidation-reduction of the electron mediator potassium ferrocyanide on the electrode surface. The catalytic reduction current of lauroyl peroxide of different concentrations on the HRP enzyme electrode was measured by chronoamperometry, a calibration curve between the catalytic reduction current and the concentration of peroxide was established accordingly, and the apparent Michaelis constant Km of the HRP enzyme in this system is determined according to Lineweaver-Burk equation.
2. The research examined that the electrode reaction process of iodine ion and chitosan on the surface of the graphite electrode belongs to the adsorptive stripping voltammetric process. For detailed process, the new material from iodine ions combine with protonized chitosan enriched on the electrode surface. Through the potential scan from negative to positive, due to lose electrons on the surface of the electrode, iodine ions is oxidized to the free iodine elemental which cannot be chitosan adsorption, and then stripping out from the electrode surface, at the same time produce the oxidation peak current. The best determination of iodine ion conditions are:0.1mol/L-citric acid sodium citrate buffer (pH5.0) as the base solution, chitosan concentration is200mu g/mL; Adsorptive stripping parameters:the enriched potential0v, the enriched time180s, the static potential0v,the static time25s; The scanning parameters of differential pulse voltammetry:beginning potential0.1V, terminate potential1.5V, potential increment0.004V, amplitude0.05V, pulse width0.1s, pulse cycle0.2s; In this condition, the iodine ion concentration producing by the oxidation peak current of graphite electrode is in range of0.07～0.49g/L, and show good linear correlation (R=0.9990). This method has a certain anti-interference ability, and show good reproducibility and stability. The used electrodes can be repeated use by some renewable activation steps.
3. In the present study, mechanism of the chitosan modified screen-printed electrode catalytic reac tion was described and experimental conditions were defined for the determination of peroxide value i n vegetable oil using differential pulse stripping voltammetry. Due to the fact that protonated chitosan c an absorb only iodide ion, chitosan modified electrode was applied to determine the oxidation process o f iodide. Results showed that the peak current value decreased with increasing peroxide value in the veg etable oil and reveled a good correlation (R=0.9914) with the peroxide in the concentration range of0.01～3.2μg/mL. Moreover, the peroxide value measured in the vegetable oil using chitosan modified elect rode was in agreement with the results obtained using standard iodometric method.
4. A New method in the determination of peroxide value was developed, namely by differential pulse anodic stripping voltammetry and using chitosan modified screen printing electrode scan vegetable oil samples, instead of titration to determine changes of the iodine ion concentration. In this research, the proposed method determine peroxide value of40saled vegetable oil samples, and compare with the national standard method (GB/T5538-2005). Through evaluate comprehensive deviation degree and En value to show that in the qualitative evaluation the results all satisfied. In respect of quantitative judgment have33satisfied result, the overall satisfaction rate was82.5%.Especially in the range of0.031～0.090meq/kg and0.096-0.150meq/kg the result is more accurate and satisfaction rate can reach82.35%and93.33%. This research showed that compared with the national standard method, the new method in operational, security, etc. has a certain advantage, the result accuracy was recognized within the limits of certain peroxide value range.
1.采用溶胶-凝胶法构建的双层膜结构HRP酶电极,在有机相介质中能够对过氧化月桂酰产生显著地还原电流响应,作为电子介体的亚铁氰化钾能够有效的在HRP酶与电极表面传递电子,增强还原电流响应；随着过氧化月桂酰添加量的增加,HRP酶电极产生的还原电流逐渐增强,氧化电流逐渐减弱：通过对还原峰电流与扫描速度间的关系研究,证明当扫描速度较低时HRP酶电极以传质过程控制为主,即通过HRP酶传递到电极表面发生还原反应产生还原电流。当扫描速度较高时,电极的传质过程控制转弱,逐渐转为表面过程控制,此时HRP酶电极的氧化还原反应与电子介体——亚铁氰化钾在电极表面的氧化还原有关；采用计时电流法根据不同浓度的过氧化月桂酰在HRP酶电极上的催化还原电流,建立其与过氧化物浓度间的校正曲线,并根据Lineweaver-Burk方程,确定该体系下HRP酶的表观米氏常数Km。
2.考察了碘离子与壳聚糖在石墨电极表面的电极反应过程属吸附溶出伏安过程,即碘离子与质子化的壳聚糖结合,生成的新物质在电极表面得到富集,通过由负向正的电位扫描,电极表面的碘离子失去电子,被氧化成游离的碘单质,无法被壳聚糖吸附,进而从电极表面脱附出来,同时产生氧化峰电流；最佳的碘离子测定条件为：以0.1mol/L柠檬酸-柠檬酸钠缓冲液(pH5.0)为底液,壳聚糖浓度200μg/mL;吸附溶出参数：富集电位OV,富集时间180s,而静止时间、静止电位对峰电流的影响较小,这里采用的是静止电位OV,静止时间25s；微分脉冲伏安法扫描参数：开始电位0.1V,终止电位1.5V,电位增量0.004V,振幅0.05V,脉冲宽度0.1s,脉冲周期0.2s；在此条件下,石墨电极产生的氧化峰电流与碘离子浓度在0.07-0.49g/L内,呈良好的线性相关关系(R=0.9990)。该方法具有一定的抗干扰能力,表现出良好的重现性及稳定性。使用后的电极经过一定的再生活化步骤即可重复使用。
3.阐述了壳聚糖修饰丝网印刷电极催化反应机理,确定了采用微分脉冲溶出伏安法测定植物油过氧化值的条件。由于质子化的壳聚糖只能吸附碘离子,因此壳聚糖修饰电极可以用来确定碘离子的氧化过程。结果表明,峰电流值随植物油过氧化值的升高而降低,且在0.01-3.21μg/mL范围内呈良好的相关关系(R=0.9914)。此外,将采用壳聚糖修饰电极测定得到的过氧化值与标准碘量法的测定结果进行初步比对,结果基本一致。
4.课题组研究设计一种检测过氧化值的新方法,即采用壳聚糖修饰丝网印刷电极经微分脉冲溶出伏安法扫描,来取代碘量法中人工滴定确定碘离子浓度变化的步骤。本研究采用上述方法对40种市售植物油的过氧化值进行测定,并与国标法(GB/T5538-2005)的测定结果进行比对,综合偏离度与En数两项指标进行评价,结果表明：两种方法在定性判定方面结果一致,而在定量判断方面有33个结果满意,整体满意率为82.5%,其中0.031～0.090meq/kg及0.096～0.150meq/kg区间内的结果更为准确,满意率分别为82.35%及93.33%。研究表明,与国标法相比,新方法在操作性、安全性等方面存在一定优势,在一定过氧化值范围内,结果准确性得到认可。
This research choosed peroxide value of vegetable oil as detection index, used the biologic al and chemical modification method to make two kinds of electrodes respectively, studied their electrochemical behavior in organic phase medium, discussed the reaction mechanism and the electrod e reaction process, and determined the best technical parameters. By these parameters, a good catalytic r eaction micro environment was built to meet the practical requirement of vegetable oil testing. In the me antime. this method was applied to the micro-electrodes (screen-printed electrodes), and detection tech nology and equipment which can quantify the vegetable oil quickly and accurately were developed. It s main contents are as follows:
1. The HRP enzyme electrode with a double-film structure constructed by using the Sol-Gel method can generate a significant reduction current response to lauroyl peroxide in an organic phase medium, and potassium ferrocyanide as the electron mediator can effectively transfer electrons between the HRP enzyme and the electrode surface, and enhance reduction current responses. As the added amount of lauroyl peroxide increased, the reduction current generated by the HRP enzyme electrode gradually increased, and the oxidation current gradually weakened; Through exploration of the relationship between the reduction peak current and the scanning speed, it is demonstrated that when the scanning speed is low, the HRP enzyme electrode mainly involves the mass transfer process control, that is, the reduction reaction is carried out by transfer of the HRP enzyme onto the electrode surface to generate reduction current. When the scanning speed is high, the mass transfer process control of the electrode weakens and is gradually turned into the surface process control, in which the oxidation-reduction reaction of the HRP enzyme electrode is relevant to the oxidation-reduction of the electron mediator potassium ferrocyanide on the electrode surface. The catalytic reduction current of lauroyl peroxide of different concentrations on the HRP enzyme electrode was measured by chronoamperometry, a calibration curve between the catalytic reduction current and the concentration of peroxide was established accordingly, and the apparent Michaelis constant Km of the HRP enzyme in this system is determined according to Lineweaver-Burk equation.
2. The research examined that the electrode reaction process of iodine ion and chitosan on the surface of the graphite electrode belongs to the adsorptive stripping voltammetric process. For detailed process, the new material from iodine ions combine with protonized chitosan enriched on the electrode surface. Through the potential scan from negative to positive, due to lose electrons on the surface of the electrode, iodine ions is oxidized to the free iodine elemental which cannot be chitosan adsorption, and then stripping out from the electrode surface, at the same time produce the oxidation peak current. The best determination of iodine ion conditions are:0.1mol/L-citric acid sodium citrate buffer (pH5.0) as the base solution, chitosan concentration is200mu g/mL; Adsorptive stripping parameters:the enriched potential0v, the enriched time180s, the static potential0v,the static time25s; The scanning parameters of differential pulse voltammetry:beginning potential0.1V, terminate potential1.5V, potential increment0.004V, amplitude0.05V, pulse width0.1s, pulse cycle0.2s; In this condition, the iodine ion concentration producing by the oxidation peak current of graphite electrode is in range of0.07～0.49g/L, and show good linear correlation (R=0.9990). This method has a certain anti-interference ability, and show good reproducibility and stability. The used electrodes can be repeated use by some renewable activation steps.
3. In the present study, mechanism of the chitosan modified screen-printed electrode catalytic reac tion was described and experimental conditions were defined for the determination of peroxide value i n vegetable oil using differential pulse stripping voltammetry. Due to the fact that protonated chitosan c an absorb only iodide ion, chitosan modified electrode was applied to determine the oxidation process o f iodide. Results showed that the peak current value decreased with increasing peroxide value in the veg etable oil and reveled a good correlation (R=0.9914) with the peroxide in the concentration range of0.01～3.2μg/mL. Moreover, the peroxide value measured in the vegetable oil using chitosan modified elect rode was in agreement with the results obtained using standard iodometric method.
4. A New method in the determination of peroxide value was developed, namely by differential pulse anodic stripping voltammetry and using chitosan modified screen printing electrode scan vegetable oil samples, instead of titration to determine changes of the iodine ion concentration. In this research, the proposed method determine peroxide value of40saled vegetable oil samples, and compare with the national standard method (GB/T5538-2005). Through evaluate comprehensive deviation degree and En value to show that in the qualitative evaluation the results all satisfied. In respect of quantitative judgment have33satisfied result, the overall satisfaction rate was82.5%.Especially in the range of0.031～0.090meq/kg and0.096-0.150meq/kg the result is more accurate and satisfaction rate can reach82.35%and93.33%. This research showed that compared with the national standard method, the new method in operational, security, etc. has a certain advantage, the result accuracy was recognized within the limits of certain peroxide value range.
引文
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