基于移动反应界面的电泳中和滴定新原理和新方法
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摘要
近年来,移动反应界面(Moving Reaction Boundary,MRB)的理论与实验研究发展迅速。在分析化学领域中,应用MRB理论,在毛细管电泳(CE)上分离和富集各种两性物质的研究取得了很大的进展,最大可达到对分析物上百万倍的富集,极大地解决了CE中进样量低的问题。同时,由移动中和界面(Moving Neutralization Boundary, MNB)理论推导出的判别式,用来判断界面移动方向并且为等电聚焦动力学的研究提供了新的视角。本论文的研究工作主要提出—种新的酸碱滴定方法—电迁移酸碱滴定(Electromigration Acid-Base Titration,EABT),在MNB理论基础上设计电迁移酸碱滴定的装置及其应用于强酸强碱和强碱弱酸中和滴定测定未知酸碱的浓度。
具体研究内容如下：
1、电迁移酸碱中和滴定装置
在本论文的第二章中,设计了一种用于电迁移酸碱滴定的装置,该装置由连有阳极电泳槽(酸溶液)和阴极电泳槽(碱溶液)的大管施加电压后氢离子、氢氧根离子在电压的作用下分别向负极、正极移动产生电迁移酸碱中和反应。用蠕动泵分别将阳极电泳槽和阴极电泳槽中的酸碱溶液泵入到大管中,大管的两端连有电极从而可以记录下实验中所施加的电压。在大管的正下方有一把带有标准刻度的标尺,并且在大管上方固定CCD相机用来记录移动中和反应界面的移动距离(D),根据D和浓度作出标准曲线,进而计算有关酸或碱浓度。该发明提出一种新的酸碱中和滴定方法用来测定未知酸碱浓度。本发明可应用于强酸强碱滴定/弱酸强碱滴定/蛋白质含氮量的分析。
2、强酸(HCl)强碱(NaOH)电迁移酸碱滴定
在本论文的第三章中,应用第二章设计的电迁移酸碱滴定装置,第一次设计用MNB理论进行电迁移酸碱滴定,本章用HCl、NaOH为例来进行强酸强碱电迁移酸碱滴定。实验结果显示：(1)实验中所用的琼脂糖浓度、电压和背景电解质(KCl)浓度对MNB有明显的影响。(2)在固定酸碱浓度条件下,移动反应界面的距离和反应时间成一定线性关系(3)在优化条件下,相同时间和NaOH浓度,移动中和界面的距离和自然对数的不同HCl浓度成线性关系,用线性曲线可以测定未知酸浓度。(4)实验验证了不同酸碱的指示剂对界面移动距离无明显影响从而可以避免了传统酸碱中和滴定中指示剂选择所带来的误差。(5)日内差和日间差分别少于3.76%和1.59%,表示有很好的准确度和稳定性。
3、弱酸(HAc)强碱(NaOH)电迁移酸碱滴定
在本论文的第四章中,在第三章HCl(aq)-NaOH(aq)滴定的基础上,进一步进行NaOH(aq)滴定HAc(aq)的电迁移酸碱滴定,实验结果和本文第三章得出的结果相近。(1)实验中所用的琼脂糖浓度、电压和背景电解质(KCl)浓度对MNB有明显的影响,即对EABT也有影响。(2)在固定HAc和NaOH浓度条件下,移动反应界面的距离(D)和反应时间(t)成一定线性关系,充分说明MNB的迁移速度是匀速运动。(3)在优化条件下,迁移时间(t)和NaOH浓度一定的条件下,移动中和界面距离(D)和不同HAc浓度成线性。(4)实验验证了不同酸碱的指示剂对D无明显影响从而可以避免了传统酸碱中和滴定中指示剂选择所带来的误差。(5)日内差和日间差分别少于3.54%和3.81%,表示有很好的准确度和稳定性。
电迁移酸碱滴定是在一系列已有的酸碱滴定基础上提出的,是一种全新想法的酸碱滴定,在原来已有的酸碱滴定的方法上补充了另外一种可用于酸碱滴定的新方法。
In resent years, Moving Reaction Boundary (MRB) theoretical and experimental study is developing rapidly. The concept of MRB is very useful in the field of electrophoresis. For example, the dynamic theory of isoelectric focusing (IEF) has been well developed from the concept of MNB. Second, the method of MNB has been used for the sample stacking of zwitterions (viz., proteins, or peptides, or amino acids) and drugs in capillary electrophoresis (CE) and condensation of target compound in free-flow electrophoresis (FFE). Third, the concept of MCB has been applied for the illumination on mechanism of EDTA-based sample sweeping of heavy metal ions in CE.
This paper for the first time shows that the concept of MRB can be used to design a novel acid-base titration of EABT via the MNB formed with H+ and OH-For the purpose of simplicity, we use HC1 and NaOH, HAc and NaOH as the model reactant and reagent, respectively, for the studies. Below are the relevant experiments and advantages of EABT. The main points of this thesis are summarized as follows:
1. The equipment of the EABT
In the Chapter 2, The apparatus used for the experiment of EABT is connected a glass tube which connected with the anode electrophoresis slots (acid solution) and cathodic electrophoretic slots (bace solution). Under the electric field, the hydrogen and hydroxyl ions moved in the opposite direction and had an electromigration reaction with each other. The reaction led to the formation of MNB. The tube coupled with a ruler was set on a white light plate, an adjustable digital camera was fixed over the flat plate. The digital camera could be well used to record the boundary movement. The two ends of the glass tube were connected to two three-way-pipes via the two rubber-tubes. The two pipes were further joined with two peristaltic pumps and the two vials of anode and cathode. The two pumps were used for the flows of anolyte and catholyte. A power supply was used to yield the direct electric field for the experiment of EABT.
2. Equivalence-Point Electromigration strong Acid-strong Base Titration via Moving Neutralization Boundary Electrophoresis
In the Chapter 3, we developed a novel method of acid-base titration, viz., the electromigration acid-base titration (EABT), via the apparatus of EABT was described in Chapter 2. With HCl and NaOH as the model strong acid and base, respectively, we conducted the experiments on the EABT via the method of MNR for the first time. The experiments revealed that (1) the concentration of agarose gel, the voltage used and the content of background electrolyte (KCl) had evident influence on the boundary movement; (2) the movement length was as a function of the running time under the constant acid and base concentrations; and (3) there was a good linearity between the length and natural logarithmic concentration of HCl under the optimized conditions, and the linearity could be used to detect the concentration of acid. The experiments further manifested that (1) the RSD values of intra-day and inter-day runs were less than 1.59% and 3.76%, respectively, indicating good precision and stability; (2) the indicators with different pKa values had no obvious effect on EABT, distinguishing strong influence on the judgment of equivalence-point titration in the classic one; and (3) the constant equivalence-point titration was always existed in the EABT, rather than the classic volumetric analysis. Additionally, the EABT could be well used for the determination of actual acid concentrations. The experimental results achieved herein showed a new general guidance for the development of classic volumetric analysis and element (e.g., nitrogen) analysis in protein chemistry.
3. Equivalence-Point Electromigration weak Acid-strong Base Titration via Moving Neutralization Boundary Electrophoresis
In the Chapter 4, we used HAc and NaOH as the model weak acid and strong base, respectively via the EABT. The experiments revealed that (1) the concentration of agarose gel, the voltage used and the content of background electrolyte (KC1) had evident influence on the boundary movement; (2) the movement length was as a function of the running time under the constant acid and base concentrations; and (3) there was a good linearity between the length and concentration of HAc under the optimized conditions, and the linearity could be used to detect the concentration of acid. (4) the indicators with different pKa values had no obvious effect on EABT, distinguishing strong influence on the judgment of equivalence-point titration in the classic one.
具体研究内容如下：
1、电迁移酸碱中和滴定装置
在本论文的第二章中,设计了一种用于电迁移酸碱滴定的装置,该装置由连有阳极电泳槽(酸溶液)和阴极电泳槽(碱溶液)的大管施加电压后氢离子、氢氧根离子在电压的作用下分别向负极、正极移动产生电迁移酸碱中和反应。用蠕动泵分别将阳极电泳槽和阴极电泳槽中的酸碱溶液泵入到大管中,大管的两端连有电极从而可以记录下实验中所施加的电压。在大管的正下方有一把带有标准刻度的标尺,并且在大管上方固定CCD相机用来记录移动中和反应界面的移动距离(D),根据D和浓度作出标准曲线,进而计算有关酸或碱浓度。该发明提出一种新的酸碱中和滴定方法用来测定未知酸碱浓度。本发明可应用于强酸强碱滴定/弱酸强碱滴定/蛋白质含氮量的分析。
2、强酸(HCl)强碱(NaOH)电迁移酸碱滴定
在本论文的第三章中,应用第二章设计的电迁移酸碱滴定装置,第一次设计用MNB理论进行电迁移酸碱滴定,本章用HCl、NaOH为例来进行强酸强碱电迁移酸碱滴定。实验结果显示：(1)实验中所用的琼脂糖浓度、电压和背景电解质(KCl)浓度对MNB有明显的影响。(2)在固定酸碱浓度条件下,移动反应界面的距离和反应时间成一定线性关系(3)在优化条件下,相同时间和NaOH浓度,移动中和界面的距离和自然对数的不同HCl浓度成线性关系,用线性曲线可以测定未知酸浓度。(4)实验验证了不同酸碱的指示剂对界面移动距离无明显影响从而可以避免了传统酸碱中和滴定中指示剂选择所带来的误差。(5)日内差和日间差分别少于3.76%和1.59%,表示有很好的准确度和稳定性。
3、弱酸(HAc)强碱(NaOH)电迁移酸碱滴定
在本论文的第四章中,在第三章HCl(aq)-NaOH(aq)滴定的基础上,进一步进行NaOH(aq)滴定HAc(aq)的电迁移酸碱滴定,实验结果和本文第三章得出的结果相近。(1)实验中所用的琼脂糖浓度、电压和背景电解质(KCl)浓度对MNB有明显的影响,即对EABT也有影响。(2)在固定HAc和NaOH浓度条件下,移动反应界面的距离(D)和反应时间(t)成一定线性关系,充分说明MNB的迁移速度是匀速运动。(3)在优化条件下,迁移时间(t)和NaOH浓度一定的条件下,移动中和界面距离(D)和不同HAc浓度成线性。(4)实验验证了不同酸碱的指示剂对D无明显影响从而可以避免了传统酸碱中和滴定中指示剂选择所带来的误差。(5)日内差和日间差分别少于3.54%和3.81%,表示有很好的准确度和稳定性。
电迁移酸碱滴定是在一系列已有的酸碱滴定基础上提出的,是一种全新想法的酸碱滴定,在原来已有的酸碱滴定的方法上补充了另外一种可用于酸碱滴定的新方法。
In resent years, Moving Reaction Boundary (MRB) theoretical and experimental study is developing rapidly. The concept of MRB is very useful in the field of electrophoresis. For example, the dynamic theory of isoelectric focusing (IEF) has been well developed from the concept of MNB. Second, the method of MNB has been used for the sample stacking of zwitterions (viz., proteins, or peptides, or amino acids) and drugs in capillary electrophoresis (CE) and condensation of target compound in free-flow electrophoresis (FFE). Third, the concept of MCB has been applied for the illumination on mechanism of EDTA-based sample sweeping of heavy metal ions in CE.
This paper for the first time shows that the concept of MRB can be used to design a novel acid-base titration of EABT via the MNB formed with H+ and OH-For the purpose of simplicity, we use HC1 and NaOH, HAc and NaOH as the model reactant and reagent, respectively, for the studies. Below are the relevant experiments and advantages of EABT. The main points of this thesis are summarized as follows:
1. The equipment of the EABT
In the Chapter 2, The apparatus used for the experiment of EABT is connected a glass tube which connected with the anode electrophoresis slots (acid solution) and cathodic electrophoretic slots (bace solution). Under the electric field, the hydrogen and hydroxyl ions moved in the opposite direction and had an electromigration reaction with each other. The reaction led to the formation of MNB. The tube coupled with a ruler was set on a white light plate, an adjustable digital camera was fixed over the flat plate. The digital camera could be well used to record the boundary movement. The two ends of the glass tube were connected to two three-way-pipes via the two rubber-tubes. The two pipes were further joined with two peristaltic pumps and the two vials of anode and cathode. The two pumps were used for the flows of anolyte and catholyte. A power supply was used to yield the direct electric field for the experiment of EABT.
2. Equivalence-Point Electromigration strong Acid-strong Base Titration via Moving Neutralization Boundary Electrophoresis
In the Chapter 3, we developed a novel method of acid-base titration, viz., the electromigration acid-base titration (EABT), via the apparatus of EABT was described in Chapter 2. With HCl and NaOH as the model strong acid and base, respectively, we conducted the experiments on the EABT via the method of MNR for the first time. The experiments revealed that (1) the concentration of agarose gel, the voltage used and the content of background electrolyte (KCl) had evident influence on the boundary movement; (2) the movement length was as a function of the running time under the constant acid and base concentrations; and (3) there was a good linearity between the length and natural logarithmic concentration of HCl under the optimized conditions, and the linearity could be used to detect the concentration of acid. The experiments further manifested that (1) the RSD values of intra-day and inter-day runs were less than 1.59% and 3.76%, respectively, indicating good precision and stability; (2) the indicators with different pKa values had no obvious effect on EABT, distinguishing strong influence on the judgment of equivalence-point titration in the classic one; and (3) the constant equivalence-point titration was always existed in the EABT, rather than the classic volumetric analysis. Additionally, the EABT could be well used for the determination of actual acid concentrations. The experimental results achieved herein showed a new general guidance for the development of classic volumetric analysis and element (e.g., nitrogen) analysis in protein chemistry.
3. Equivalence-Point Electromigration weak Acid-strong Base Titration via Moving Neutralization Boundary Electrophoresis
In the Chapter 4, we used HAc and NaOH as the model weak acid and strong base, respectively via the EABT. The experiments revealed that (1) the concentration of agarose gel, the voltage used and the content of background electrolyte (KC1) had evident influence on the boundary movement; (2) the movement length was as a function of the running time under the constant acid and base concentrations; and (3) there was a good linearity between the length and concentration of HAc under the optimized conditions, and the linearity could be used to detect the concentration of acid. (4) the indicators with different pKa values had no obvious effect on EABT, distinguishing strong influence on the judgment of equivalence-point titration in the classic one.
引文
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