核黄素结晶分离纯化研究
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摘要
本文主要研究了核黄素结晶分离纯化相关问题。首先,采用平衡法测定了核黄素在纯水和在283K、288K、293K、298K及303K的HCl-水体系(HCl的摩尔分率w分别为0.14,0.16,0.17,0.20,0.24)中的溶解度,用溶解度模型-λh方程回归了上述溶解度数据,λh方程的关联效果在整个溶剂浓度变化区间内很稳定,收敛性较好,并回归得到模型参数λ和h与w的经验关系式;测定核黄素在283K,288K和293K的HCl-水体系中溶解与超溶解特性,得到核黄素的结晶介稳区,这些热力学研究为核黄素的工业化生产奠定基础。在主溶剂、析出剂、温度等单因素实验基础上,应用多因素二水平实验经方差分析筛选显著性因子,并由SAS软件(Statistic Analysis System)响应面优化程序研究核黄素溶析结晶过程及其参数控制。
第二部分研究物理场对结晶的影响,稳定核黄素球状晶习。核黄素固体粉末及其乙醇-盐酸溶液的顺磁共振波谱测定,说明核黄素分子中有未配对电子,具有顺磁性。从结晶动力学角度探讨超声溶析结晶机理,超声作用不仅可以加快成核速率、缩短诱导期,加快结晶进程,使晶体粒度均匀,稳定球状晶习,且不影响核黄素的纯度和回收率。
第三部分针对Bacillus subtilis 24/pMX45核黄素发酵液的特性,比较几种发酵液处理方法的优缺点。结果表明:直接处理法所得核黄素样品杂质含量高;碱溶法得到收率为55%,纯度为92.5%针状晶习核黄素。控制结晶法结合超声溶析结晶提取纯化方法,可得纯度为98.3%、收率为73%的核黄素球状结晶,且其主要质量指标符合GB14752-93和2005年版中华人民共和国药典要求,并且流散性提高。
最后,探讨大孔吸附树脂对核黄素的吸附性能,结果表明:AB-8大孔吸附树脂对核黄素选择吸附性较强,其静态饱和吸附量为12.1mg/g树脂;采用乙醇-0.1mol/L NaOH溶液(v/v,1:1)洗脱,最高浓度提高了将近5倍。应用AB-8大孔吸附树脂能将废液中约44%的核黄素回收。
First, the solubilities of riboflavin in pure water and in different concentration hydrochloride aqueous solution (w =0.14,0.16,0.17,0.20,0.24,‘w’meant the HCl fraction in mixture aqueous solution) under different temperatures, viz. 283K, 288K, 293K, 298K, 303K, etc., were measured by steady method, respectively. The data of solubility was obtained through regression of solubility model——the equation ofλh, which relevancy effect was stable and convergent in hydrochloride aqueous solution. The empirical formulae betweenλ, h and w were obtained. The solubility and supersolubility of riboflavin in hydrochloride aqueous solution under 283K,288K, 293K, etc. were mensurated and analyzed. The width of crystallization metastable zone was determined. These studies about crystallization thermodynamics made the base for the industrial manufacture of riboflavin. Based on single factor experiments, such as the prime solvent, diluent and temperature, etc., the significance factors were gotten from the variance analysis of the Two-Lever Experiment Design. By use of the Response-Surface Test, with the Statistic Analysis Software( SAS ), the dilution crystallization process and the controlling parameters were optimized.
Next, the effects of physical field on riboflavin crystallization were studied to obtain stable sphericity crystal. From the electron paramagnetic resonance spectrum of riboflavin, there was an un-partnership electron in riboflavin, that meant riboflavin was paramagnetism. The nucleation rate and growth rate, which were of importance for the design and performance of the industrial crystallizer, were accelerated in ultrasound field, and the crystalization inducement period was shortened. Moreover, the regular sphericity crystal grain was gotten in appropriate ultrasonic field, and riboflavin yield and purity were not affected.
The third, according to the peculiarity of Bacillus subtilis 24/pMX45 fermentation broth, serveral ways of riboflavin separation and purification were analyzed. As a result, there was high impurity in sample from the straight-separation method. The yield and purity of riboflavin product from the alkali-dissolved method were 55%, 92.5%, respectively. The crystal shape was needle. However, followed crystallization-control way, applied ultrasonic dilution crystallization processing, globe riboflavin crystal yield was 73% and purity 98.3%. Furthermore, the main quality indexes accorded with GB14752-93 and the codex of P.R.C., and the flow-scatteredness was increased.
In the end, the adsorbility of macroporous adsorptive resins for riboflavin were studied. The results showed that the saturated static adsorption of AB-8 resin was 12.1mg/g resin. And the effects of concentration、pH、temperature on the adsorbilities of AB-8 resin were measured. Meanwhile, under the conditions of pH=6 and normal temperature, the dynamic adsorption curve of AB-8 resin for riboflavin was determined. Additionally, through the elution curves, the optimal eluant was the mixture of alcohol and 0.1M NaOH (1:1, v/v). Applying AB-8 resin, about 44% remnant riboflavin in crystallization mother liquor and cleaning solution were reclaimed.
第二部分研究物理场对结晶的影响,稳定核黄素球状晶习。核黄素固体粉末及其乙醇-盐酸溶液的顺磁共振波谱测定,说明核黄素分子中有未配对电子,具有顺磁性。从结晶动力学角度探讨超声溶析结晶机理,超声作用不仅可以加快成核速率、缩短诱导期,加快结晶进程,使晶体粒度均匀,稳定球状晶习,且不影响核黄素的纯度和回收率。
第三部分针对Bacillus subtilis 24/pMX45核黄素发酵液的特性,比较几种发酵液处理方法的优缺点。结果表明:直接处理法所得核黄素样品杂质含量高;碱溶法得到收率为55%,纯度为92.5%针状晶习核黄素。控制结晶法结合超声溶析结晶提取纯化方法,可得纯度为98.3%、收率为73%的核黄素球状结晶,且其主要质量指标符合GB14752-93和2005年版中华人民共和国药典要求,并且流散性提高。
最后,探讨大孔吸附树脂对核黄素的吸附性能,结果表明:AB-8大孔吸附树脂对核黄素选择吸附性较强,其静态饱和吸附量为12.1mg/g树脂;采用乙醇-0.1mol/L NaOH溶液(v/v,1:1)洗脱,最高浓度提高了将近5倍。应用AB-8大孔吸附树脂能将废液中约44%的核黄素回收。
First, the solubilities of riboflavin in pure water and in different concentration hydrochloride aqueous solution (w =0.14,0.16,0.17,0.20,0.24,‘w’meant the HCl fraction in mixture aqueous solution) under different temperatures, viz. 283K, 288K, 293K, 298K, 303K, etc., were measured by steady method, respectively. The data of solubility was obtained through regression of solubility model——the equation ofλh, which relevancy effect was stable and convergent in hydrochloride aqueous solution. The empirical formulae betweenλ, h and w were obtained. The solubility and supersolubility of riboflavin in hydrochloride aqueous solution under 283K,288K, 293K, etc. were mensurated and analyzed. The width of crystallization metastable zone was determined. These studies about crystallization thermodynamics made the base for the industrial manufacture of riboflavin. Based on single factor experiments, such as the prime solvent, diluent and temperature, etc., the significance factors were gotten from the variance analysis of the Two-Lever Experiment Design. By use of the Response-Surface Test, with the Statistic Analysis Software( SAS ), the dilution crystallization process and the controlling parameters were optimized.
Next, the effects of physical field on riboflavin crystallization were studied to obtain stable sphericity crystal. From the electron paramagnetic resonance spectrum of riboflavin, there was an un-partnership electron in riboflavin, that meant riboflavin was paramagnetism. The nucleation rate and growth rate, which were of importance for the design and performance of the industrial crystallizer, were accelerated in ultrasound field, and the crystalization inducement period was shortened. Moreover, the regular sphericity crystal grain was gotten in appropriate ultrasonic field, and riboflavin yield and purity were not affected.
The third, according to the peculiarity of Bacillus subtilis 24/pMX45 fermentation broth, serveral ways of riboflavin separation and purification were analyzed. As a result, there was high impurity in sample from the straight-separation method. The yield and purity of riboflavin product from the alkali-dissolved method were 55%, 92.5%, respectively. The crystal shape was needle. However, followed crystallization-control way, applied ultrasonic dilution crystallization processing, globe riboflavin crystal yield was 73% and purity 98.3%. Furthermore, the main quality indexes accorded with GB14752-93 and the codex of P.R.C., and the flow-scatteredness was increased.
In the end, the adsorbility of macroporous adsorptive resins for riboflavin were studied. The results showed that the saturated static adsorption of AB-8 resin was 12.1mg/g resin. And the effects of concentration、pH、temperature on the adsorbilities of AB-8 resin were measured. Meanwhile, under the conditions of pH=6 and normal temperature, the dynamic adsorption curve of AB-8 resin for riboflavin was determined. Additionally, through the elution curves, the optimal eluant was the mixture of alcohol and 0.1M NaOH (1:1, v/v). Applying AB-8 resin, about 44% remnant riboflavin in crystallization mother liquor and cleaning solution were reclaimed.
引文
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