阿斯巴甜母液中苯丙氨酸和天冬氨酸的分离
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摘要
苯丙氨酸和天冬氨酸是阿斯巴甜母液中的二种氨基酸,其中苯丙氨酸的价格很高,但是常规的分离方法无法实现这一部分苯丙氨酸清洁化的回收。本文通过选用了色谱法对苯丙氨酸和天冬氨酸进行了有效的分离,得到结果如下:
1.比较研究了紫外双波长检测法和紫外旋光混合检测法两种方法,选择了紫外双波长检测法作为苯丙氨酸和天冬氨酸的定量方法,在λ=257nm和λ=230nm波长下苯丙氨酸和天冬氨酸能准确定量,其中苯丙氨酸浓度的标准偏差<1%,天冬氨酸浓度的标准偏差<3%。
2.论文选用了色谱法作为苯丙氨酸和天冬氨酸的分离方法,以水作流动相,苯丙氨酸和天冬氨酸在吸附树脂上表现出了良好的分离效果。进一步研究了树脂的极性、比表面积和孔容对苯丙氨酸和天冬氨酸的色谱分离的影响,其中树脂的极性起了决定性作用,苯丙氨酸和天冬氨酸在非极性和弱极性吸附树脂上普遍能实现分离,以AB-8树脂作分离填料时,分离度达到了6.887。
苯丙氨酸在吸附树脂上的的保留时间大小顺序:弱极性树脂>非极性树脂>中极性树脂>极性树脂;天冬氨酸的保留时间随树脂极性的增加逐步增加。在以交联聚苯乙烯为骨架的弱极性吸附树脂上,天冬氨酸的保留时间随孔容的增加逐步增加,随树脂的比表面积增加而减小,苯丙氨酸的保留时间随着树脂比表面积和孔容的改变,出现小范围内的波动。
3.苯丙氨酸和AB-8树脂之间同时存在疏水作用氢键作用,天冬氨酸与AB-8树脂之间存在氢键作用。氢键作用是实现苯丙氨酸和天冬氨酸在AB-8树脂上分离的关键,在整个色谱分离过程中占主导。以水为流动相,疏水作用拉近了苯丙氨酸分子疏水部分与树脂表面的氢键作用位点的距离,促进氢键作用形成。
洗脱剂中盐的存在,减弱了水分子对苯丙氨酸疏水基团的保护作用,使得苯丙氨酸分子与AB-8树脂间疏水作用增强。在较低盐浓度条件下,苯丙氨酸的保留时间几乎成线性增加。在极低盐浓度下,疏水作用的增强必须克服某一个阀值才能使得氢键作用增强,这种增强作用随离子强度增加瞬时完成,体现为苯丙氨酸保留时间的陡然增加。当盐的离子强度进一步增加时,氢键作用的增强幅度不再发生变化。
4.论文采用示踪剂法,测得分析柱的总空隙率和外空隙率分别为0.790和0.310。然后选用了前沿分析法测定了苯丙氨酸和天冬氨酸吸附等温线。研究Asp存在的情况下Phe在AB-8上的吸附行为,结果显示AB-8树脂上存在2个吸附位点,Asp的存在不会对Phe所占据的吸附位点形成竞争。
There are two kinds of amino acids (phenylalanine and aspartic acid) existing in the aspartame mother liquor, phenylalanine couldn’t be reclaimed by the routine separation methods, which is very expensive in the market. Chromatographic separation method was selected in the separation process of phenylalanine and aspartic acid which could separate them from each other. The results of this thesis were listed as follow:
1. Compared with the other examination methods, the double-wavelength ultraviolet examination was applied, which the concentration of this two amino acids can be easily and extremely accurate quantified at the wavelength 257nm and 230nm. It also calculated the standard deviation of this method when it was applied in industry, The standard deviation of phenylalanine and aspartic acid were less than 1% and 3% respectively.
2. The chromatographic separation was selected in this thesis. Several different kinds of representative stationary phase and mobile phase were selected in the separation of phenylalanine and aspartic acid. The result indicated that the adsorption resins were well behaved when water was used as the mobile phase. Then the three important characters of adsorption resins were studied in further at the same chromatographic conditions. It was revealed that polarity of the adsorption resins was the key ingredient in the whole process. Phenylalanine and aspartic acid could be effective separated on the non-polar and weak-polar resins, the separation degree on AB-8 can reach to 6.887.
The retention time order of phenylalanine on adsorption resins were as follow: weak-polarity>non-polarity>middle-polarity>polarity resins; the retention time of aspartic acid were increased with the polarity increase, and it also increased with the prove volume increase, but not for the surface area change on the weak-polarity adsorption resins. The retention time of phenylalanine were fluctuated in a small range with the change of surface area and prove volume of weak-polarity adsorption resins
3. There were two kinds of acting force existing in the adsorption process, hydrophobic interaction and hydrogen bonding interaction, while only hydrogen bonding interaction existed in the adsorption of aspartic acid on Ab-8 resin. The hydrogen bonding played a predominant role in the separation process, and the hydrophobic interaction would shorten the distance between the two hydrophobic groups of AB-8 resin and phenylalanine, which would strengthen the hydrogen bonding interaction consequently in water. The hydrogen bonding interaction would be strengthened by adding some salt in water. Because the surface tension of the water could be decreased, which weaken the protection of the hydrophobic groups by water and promoted to the integration of the hydrophobic groups.
When the ion strength contunine to decrease, the result indicated that the retention time of phenylalanine increased in linear with the ionic strength, and it also revealed that the strengthen of the hydrogen bonding interaction didn’t appear until conquer a fixed value, then it will complete instantaneously.
4. The value of the total and external voidage were 0.790 and 0.310 respectively, which were investigated by radiotracer method. The adsorption isotherm of phenylalanine and aspartic acids were investaigated by the frontal analysis. Then the adsorption behavior of phenylalanine was studied when aspartic acids existing in the mixture, it was indicated that there were two adsorption points on AB-8 resin, and there was no competition between phenylalanine and aspartic acids on AB-8 resin.
1.比较研究了紫外双波长检测法和紫外旋光混合检测法两种方法,选择了紫外双波长检测法作为苯丙氨酸和天冬氨酸的定量方法,在λ=257nm和λ=230nm波长下苯丙氨酸和天冬氨酸能准确定量,其中苯丙氨酸浓度的标准偏差<1%,天冬氨酸浓度的标准偏差<3%。
2.论文选用了色谱法作为苯丙氨酸和天冬氨酸的分离方法,以水作流动相,苯丙氨酸和天冬氨酸在吸附树脂上表现出了良好的分离效果。进一步研究了树脂的极性、比表面积和孔容对苯丙氨酸和天冬氨酸的色谱分离的影响,其中树脂的极性起了决定性作用,苯丙氨酸和天冬氨酸在非极性和弱极性吸附树脂上普遍能实现分离,以AB-8树脂作分离填料时,分离度达到了6.887。
苯丙氨酸在吸附树脂上的的保留时间大小顺序:弱极性树脂>非极性树脂>中极性树脂>极性树脂;天冬氨酸的保留时间随树脂极性的增加逐步增加。在以交联聚苯乙烯为骨架的弱极性吸附树脂上,天冬氨酸的保留时间随孔容的增加逐步增加,随树脂的比表面积增加而减小,苯丙氨酸的保留时间随着树脂比表面积和孔容的改变,出现小范围内的波动。
3.苯丙氨酸和AB-8树脂之间同时存在疏水作用氢键作用,天冬氨酸与AB-8树脂之间存在氢键作用。氢键作用是实现苯丙氨酸和天冬氨酸在AB-8树脂上分离的关键,在整个色谱分离过程中占主导。以水为流动相,疏水作用拉近了苯丙氨酸分子疏水部分与树脂表面的氢键作用位点的距离,促进氢键作用形成。
洗脱剂中盐的存在,减弱了水分子对苯丙氨酸疏水基团的保护作用,使得苯丙氨酸分子与AB-8树脂间疏水作用增强。在较低盐浓度条件下,苯丙氨酸的保留时间几乎成线性增加。在极低盐浓度下,疏水作用的增强必须克服某一个阀值才能使得氢键作用增强,这种增强作用随离子强度增加瞬时完成,体现为苯丙氨酸保留时间的陡然增加。当盐的离子强度进一步增加时,氢键作用的增强幅度不再发生变化。
4.论文采用示踪剂法,测得分析柱的总空隙率和外空隙率分别为0.790和0.310。然后选用了前沿分析法测定了苯丙氨酸和天冬氨酸吸附等温线。研究Asp存在的情况下Phe在AB-8上的吸附行为,结果显示AB-8树脂上存在2个吸附位点,Asp的存在不会对Phe所占据的吸附位点形成竞争。
There are two kinds of amino acids (phenylalanine and aspartic acid) existing in the aspartame mother liquor, phenylalanine couldn’t be reclaimed by the routine separation methods, which is very expensive in the market. Chromatographic separation method was selected in the separation process of phenylalanine and aspartic acid which could separate them from each other. The results of this thesis were listed as follow:
1. Compared with the other examination methods, the double-wavelength ultraviolet examination was applied, which the concentration of this two amino acids can be easily and extremely accurate quantified at the wavelength 257nm and 230nm. It also calculated the standard deviation of this method when it was applied in industry, The standard deviation of phenylalanine and aspartic acid were less than 1% and 3% respectively.
2. The chromatographic separation was selected in this thesis. Several different kinds of representative stationary phase and mobile phase were selected in the separation of phenylalanine and aspartic acid. The result indicated that the adsorption resins were well behaved when water was used as the mobile phase. Then the three important characters of adsorption resins were studied in further at the same chromatographic conditions. It was revealed that polarity of the adsorption resins was the key ingredient in the whole process. Phenylalanine and aspartic acid could be effective separated on the non-polar and weak-polar resins, the separation degree on AB-8 can reach to 6.887.
The retention time order of phenylalanine on adsorption resins were as follow: weak-polarity>non-polarity>middle-polarity>polarity resins; the retention time of aspartic acid were increased with the polarity increase, and it also increased with the prove volume increase, but not for the surface area change on the weak-polarity adsorption resins. The retention time of phenylalanine were fluctuated in a small range with the change of surface area and prove volume of weak-polarity adsorption resins
3. There were two kinds of acting force existing in the adsorption process, hydrophobic interaction and hydrogen bonding interaction, while only hydrogen bonding interaction existed in the adsorption of aspartic acid on Ab-8 resin. The hydrogen bonding played a predominant role in the separation process, and the hydrophobic interaction would shorten the distance between the two hydrophobic groups of AB-8 resin and phenylalanine, which would strengthen the hydrogen bonding interaction consequently in water. The hydrogen bonding interaction would be strengthened by adding some salt in water. Because the surface tension of the water could be decreased, which weaken the protection of the hydrophobic groups by water and promoted to the integration of the hydrophobic groups.
When the ion strength contunine to decrease, the result indicated that the retention time of phenylalanine increased in linear with the ionic strength, and it also revealed that the strengthen of the hydrogen bonding interaction didn’t appear until conquer a fixed value, then it will complete instantaneously.
4. The value of the total and external voidage were 0.790 and 0.310 respectively, which were investigated by radiotracer method. The adsorption isotherm of phenylalanine and aspartic acids were investaigated by the frontal analysis. Then the adsorption behavior of phenylalanine was studied when aspartic acids existing in the mixture, it was indicated that there were two adsorption points on AB-8 resin, and there was no competition between phenylalanine and aspartic acids on AB-8 resin.
引文
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2.张伟国,钱和,氨基酸生产技术及其应用,中国轻工业出版社,1997
3.蒋滢,氨基酸的应用,世界图书出版公司,1996
4.中国发酵工业协会,全国发酵行业新产品、新工艺、新设备、新技术展示交流会论文,2002,11
5.袁亦丞, Nutrasweet公司及阿斯巴甜,糖业食品科技,1993(2):31～32
6.蒋兴仁,阿斯巴甜简介,食品工业科技,1989(3):42～45
7.Hill John B,Process of the Preparation of Asp-L-Phe-Methy-Ester Hydrochloride by Use of Isolated N-formyl-L-Asp,USA(4,946,988),1990(08):21
8.Bachman Gerald,Process for the Preparation of A-L-Asp-L-Phe Alky-Esters, USA(3, 933, 781),1976(1):20
9.蒋明哲,章日春,张文娟,复合甜味剂中甜味素的反相高效液相色谱测定,食品科学,1996,17(6):67～69
10.镰田光雄,崛川敏治,阿斯巴甜的近况和应用现状,食品添加剂,1996(1):56～58
11.S,J,Chem,咨询有限公司,苯丙氨酸的现状与发展前景,精细与化学用品,2002(10)
12.吴月,范伟平,欧阳平凯,天冬氨酸结晶过程南,京化工大学学报,1999(28): 21～24
13.丁绪淮,谈遒,工业结晶,北京:中国化学工业出版社,1985:36～39
14.李良铸等,生化制药学,北京:中国医药科技出版社,1991
15.刘阳生等,萃取分离氨基酸研究进展,现代化工,1998(8):8～11
16.钱庭宝,离子交换剂应用技术,天津科学技术出版社,1984
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