CN115007110B - 衣康酸功能化亲水毛细管电色谱整体柱及制备方法和应用 - Google Patents
衣康酸功能化亲水毛细管电色谱整体柱及制备方法和应用 Download PDFInfo
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Abstract
本发明提出了一种衣康酸功能化亲水毛细管电色谱整体柱及制备方法和应用，所述毛细管电色谱整体柱的固定相为：衣康酸、2‑丙烯酸‑2‑羟基‑1,3‑丙二酯、催化剂过硫酸铵在PEG 400和H2O中发生原位聚合反应，生成的多孔有机骨架整体材料。本发明提出了采用衣康酸作为功能单体通过原位聚合法制备了亲水色谱性能优异的整体柱，实现了极性核苷类药物的高效、高选择性分离分析。
Description
技术领域
本发明涉及药物分析技术领域，特别是指一种衣康酸功能化亲水毛细管电色谱整体柱及制备方法和应用。
背景技术
极性物质分析一直是分离科学领域的难点，存在反相色谱不保留或保留弱、分离度低、共流出以及正相色谱保留过强导致的不易洗脱等问题。亲水毛细管电色谱是亲水色谱和毛细管电色谱的有机结合，是实现极性物质高效、高选择性分离的一种有效途径。亲水毛细管电色谱整体柱制备方法简单，通透性好，传质速率快，分离选择性高，在极性物质分析中具有广阔的应用前景，是极性物质色谱分离研究中的重要内容。随着药物分析和生命科学的快速发展，极性物质分析需求日益增加，开发新型亲水毛细管电色谱整体柱是非常有价值的。
当前，尚且没有关于采用衣康酸作为功能单体制备亲水毛细管电色谱整体柱的研究报道。此外，现有的衣康酸键合硅胶液相色谱固定相存在制备方法复杂，成本高，分辨率低，色谱峰展宽及拖尾等问题，限制了其推广应用。开展衣康酸功能化亲水有机聚合物毛细管电色谱整体柱研究可以有效解决上述问题。
发明内容
本发明目的在于解决分离科学领域强极性物质分析面临的难题，提出了一种衣康酸功能化亲水毛细管电色谱整体柱及制备方法和应用，采用衣康酸作为功能单体通过原位聚合法制备了亲水色谱性能优异的整体柱，实现了极性核苷类药物的高效、高选择性分离分析。
本发明的技术方案是这样实现的：一种衣康酸功能化亲水毛细管电色谱整体柱，所述毛细管电色谱整体柱的固定相为：衣康酸、2-丙烯酸-2-羟基-1,3-丙二酯、催化剂过硫酸铵在PEG 400和H2O中发生原位聚合反应，生成的多孔有机骨架整体材料。
进一步地，所述的固定相由以下质量百分比的组分制备：1.0％-7.0％的衣康酸，4.0％-21.0％的2-丙烯酸-2-羟基-1,3-丙二酯，40.0％-53.0％的PEG 400和24.0％-42.0％的H2O，还包括催化剂过硫酸铵，过硫酸铵的用量是衣康酸和2-丙烯酸-2-羟基-1,3-丙二酯总质量的1.0％-2.5％。
一种衣康酸功能化亲水毛细管电色谱整体柱的制备方法，包括以下步骤：
(1)对石英毛细管预处理，使毛细管内壁裸漏出硅羟基；
(2)对预处理后的毛细管内壁乙烯基化修饰；
(3)配置聚合物溶液，超声混合均匀且除去气泡，所述聚合物溶液包括以下质量百分比的组分：1.0％-7.0％的衣康酸，4.0％-21.0％的2-丙烯酸-2-羟基-1,3-丙二酯，40.0％-53.0％的PEG 400，24.0％-42.0％的H2O，还包括催化剂过硫酸铵，过硫酸铵的用量为衣康酸和2-丙烯酸-2-羟基-1,3-丙二酯总质量的1.0％-2.5％；
(4)将步骤(3)配置的均一聚合物溶液注入到步骤(2)内壁经过乙烯基化修饰的毛细管中，两端密封，55-80℃水浴反应8-20h；反应完成后，使用甲醇冲洗毛细管整体柱，除去未反应的原料，得到衣康酸功能化亲水毛细管电色谱整体柱。
进一步地，步骤(1)中，依次采用碱溶液、酸溶液依次冲洗毛细管，使毛细管内壁裸漏出硅羟基；随后用H2O、甲醇依次冲洗毛细管；最后用氮气吹干。
进一步地，所述碱溶液为NaOH溶液，酸溶液为HCl溶液。
进一步地，步骤(1)为：石英毛细管依次使用1-1.5M NaOH溶液冲洗1-2h，1-1.5MHCl溶液冲洗1-2h，超纯水冲洗1h，甲醇冲洗20min，最后用氮气吹干。
进一步地，步骤(2)中，在预处理后的毛细管中注入甲基丙烯酰氧基丙基三甲氧硅烷甲醇溶液，使用Teflon两通套管连接毛细管两端成环状，放置于40-50℃水浴锅中反应10-16h；用甲醇冲洗毛细管，除去未与毛细管内壁键合的硅烷化试剂，再用氮气吹干毛细管。
进一步地，所述甲基丙烯酰氧基丙基三甲氧硅烷甲醇溶液，其体积分数为40-50％。
所述的衣康酸功能化亲水毛细管电色谱整体柱在极性小分子药物分离分析中应用。
进一步地，所述的极性小分子药物为核苷类药物。
本发明的有益效果：
本发明制备了新型衣康酸功能化亲水有机聚合物毛细管电色谱整体柱，衣康酸带有丰富的羧基基团，亲水性强；羧基基团是离子化基团，解离后带负电荷，可在pH 4.5-12.0产生较强的电渗流，能够满足毛细管电色谱基于电渗流驱动流动相的要求，所制备整体柱具有优异的亲水色谱分离选择性能，实现了核苷类药物的快速高效分离分析。
本发明采用功能单体衣康酸与交联剂2-丙烯酸-2-羟基-1,3-丙二酯，在二元致孔剂聚乙二醇400和超纯水中发生热引发原位聚合反应，生成多孔有机骨架整体材料。本发明的衣康酸功能化有机聚合物整体柱，制备方法简单，酸碱耐受性好，亲水色谱性能优异，重现性好；可在含有低比例有机相的流动相条件下实现极性小分子药物的快速、高效分离分析，有效降低了亲水色谱中有机相的用量，绿色经济，为极性物质分析提供了新的分析技术。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例1所制备的衣康酸功能化亲水有机聚合物毛细管电色谱整体柱的横截面扫描电镜图(A 750×；B 3000×；C 10000×)。
图2为本发明实施例1所制备亲水整体柱对极性小分子核苷类药物的电色谱分离图，图中色谱峰分别为：0.甲苯、1.碘苷、2.5-甲基尿苷、3.阿糖胞苷、4.阿扎胞苷、5.环胞苷。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：
截取长35cm(内径100μm)的熔融石英毛细管，经预处理后，用注射器注入体积分数为50％(v/v)的甲基丙烯酰氧基丙基三乙氧硅烷甲醇溶液，用Teflon两通套管连接毛细管两端成环状，放置于45℃水浴锅中反应12h，对毛细管内壁进行乙烯基化修饰，然后用甲醇冲洗毛细管30min，氮气吹干待用。
将4.0％(w/w)的衣康酸，16.0％(w/w)的2-丙烯酸-2-羟基-1,3-丙二酯，48.0％(w/w)的PEG 400，32.0％(w/w)的超纯水，2.0％(w/w)的衣康酸和2-丙烯酸-2-羟基-1,3-丙二酯总质量的过硫酸铵，置于2mL eppendorf管中，超声15min混合均匀且除气泡，配制均一的聚合物溶液。
将上述聚合物溶液注入内壁经乙烯基化修饰的毛细管中，控制注入长度为25cm，65℃水浴反应18h。反应完成后，使用甲醇冲洗毛细管整体柱2h，除去未反应的单体、交联剂以及致孔剂、未与整体柱固定相键合的小分子聚合物等残留物，制备得到衣康酸功能化亲水有机聚合物固定相毛细管电色谱整体柱，用扫面电子显微镜观察其横截面形貌，如图1所示，整体柱固定相连续均匀，呈现出丰富的多孔结构。
将实施例1所制备的衣康酸功能化亲水有机聚合物毛细管整体柱用于电色谱分离，应用电压20kV，流动相：pH7.5 15mM NaH2PO4-Na2HPO4/乙腈(30/70，v/v)，甲苯为不保留标记物，成功实现了核苷类药物的基线分离，分离度大于1.55，色谱图如图2所示。分析物根据极性从弱到强依次被洗脱，出峰顺序依次为：甲苯﹤碘苷﹤5-甲基尿苷﹤阿糖胞苷﹤阿扎胞苷﹤环胞苷，符合亲水色谱保留机制。上述结果表明该整体柱具有良好的亲水色谱分离性能，在药物分析领域应用前景广阔。
实施例2：
截取长35cm(内径100μm)的熔融石英毛细管，经预处理后，用注射器注入体积分数为50％(v/v)的甲基丙烯酰氧基丙基三乙氧硅烷甲醇溶液，用Teflon两通套管连接毛细管两端成环状，放置于45℃水浴锅中反应12h，对毛细管内壁进行乙烯基化修饰，然后用甲醇冲洗毛细管30min，氮气吹干待用。
将6.0％(w/w)的衣康酸，14.0％(w/w)的2-丙烯酸-2-羟基-1,3-丙二酯，48.0％(w/w)的PEG 400，32.0％(w/w)的超纯水，2.0％(w/w)的衣康酸和2-丙烯酸-2-羟基-1,3-丙二酯总质量的过硫酸铵，置于2mL eppendorf管中，超声15min混合均匀且除气泡，配制均一的聚合物溶液。
将上述聚合物溶液注入内壁经乙烯基化修饰的毛细管中，控制注入长度为25cm，65℃水浴反应18h。反应完成后，使用甲醇冲洗毛细管整体柱2h，除去未反应的单体、交联剂以及致孔剂、未与整体柱固定相键合的小分子聚合物等残留物，制备得到衣康酸功能化亲水有机聚合物固定相毛细管电色谱整体柱。
实施例3：
截取长35cm(内径100μm)的熔融石英毛细管，经预处理后，用注射器注入体积分数为50％(v/v)的甲基丙烯酰氧基丙基三乙氧硅烷甲醇溶液，用Teflon两通套管连接毛细管两端成环状，放置于45℃水浴锅中反应12h，对毛细管内壁进行乙烯基化修饰，然后用甲醇冲洗毛细管30min，氮气吹干待用。
将5％(w/w)的衣康酸，20％(w/w)的2-丙烯酸-2-羟基-1,3-丙二酯，41.3％(w/w)的PEG 400，33.7％(w/w)的超纯水，2.0％(w/w)的衣康酸和2-丙烯酸-2-羟基-1,3-丙二酯总质量的过硫酸铵，置于2mL eppendorf管中，超声15min混合均匀且除气泡，配制均一的聚合物溶液。
将上述聚合物溶液注入内壁经乙烯基化修饰的毛细管中，控制注入长度为25cm，65℃水浴反应18h。反应完成后，使用甲醇冲洗毛细管整体柱2h，除去未反应的单体、交联剂以及致孔剂、未与整体柱固定相键合的小分子聚合物等残留物，制备得到衣康酸功能化亲水有机聚合物固定相毛细管电色谱整体柱。
实施例4：
截取长35cm(内径75μm)的熔融石英毛细管，经预处理后，用注射器注入体积分数为50％(v/v)的甲基丙烯酰氧基丙基三乙氧硅烷甲醇溶液，用Teflon两通套管连接毛细管两端成环状，放置于45℃水浴锅中反应12h，对毛细管内壁进行乙烯基化修饰，然后用甲醇冲洗毛细管30min，氮气吹干待用。
将4.0％(w/w)的衣康酸，16.0％(w/w)的2-丙烯酸-2-羟基-1,3-丙二酯，48.0％(w/w)的PEG 400，32.0％(w/w)的超纯水，2.0％(w/w)的衣康酸和2-丙烯酸-2-羟基-1,3-丙二酯总质量的过硫酸铵，置于2mL eppendorf管中，超声15min混合均匀且除气泡，配制均一的聚合物溶液。
将上述聚合物溶液注入内壁经乙烯基化修饰的毛细管中，控制注入长度为25cm，65℃水浴反应18h。反应完成后，使用甲醇冲洗毛细管整体柱2h，除去未反应的单体、交联剂以及致孔剂、未与整体柱固定相键合的小分子聚合物等残留物，制备得到衣康酸功能化亲水有机聚合物固定相毛细管电色谱整体柱。
实施例5：
截取长35cm(内径75μm)的熔融石英毛细管，经预处理后，用注射器注入体积分数为50％(v/v)的甲基丙烯酰氧基丙基三乙氧硅烷甲醇溶液，用Teflon两通套管连接毛细管两端成环状，放置于45℃水浴锅中反应12h，对毛细管内壁进行乙烯基化修饰，然后用甲醇冲洗毛细管30min，氮气吹干待用。
将7.0％(w/w)的衣康酸，21.0％(w/w)的2-丙烯酸-2-羟基-1,3-丙二酯，40.0％(w/w)的PEG 400，32.0％(w/w)的超纯水，2.5％(w/w)的衣康酸和2-丙烯酸-2-羟基-1,3-丙二酯总质量的过硫酸铵，置于2mL eppendorf管中，超声15min混合均匀且除气泡，配制均一的聚合物溶液。
将上述聚合物溶液注入内壁经乙烯基化修饰的毛细管中，控制注入长度为25cm，65℃水浴反应18h。反应完成后，使用甲醇冲洗毛细管整体柱2h，除去未反应的单体、交联剂以及致孔剂、未与整体柱固定相键合的小分子聚合物等残留物，制备得到衣康酸功能化亲水有机聚合物固定相毛细管电色谱整体柱。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种衣康酸功能化亲水毛细管电色谱整体柱，其特征在于：所述毛细管电色谱整体柱的固定相为：衣康酸、2-丙烯酸-2-羟基-1,3-丙二酯、催化剂过硫酸铵在PEG 400和H2O中发生原位聚合反应，生成的多孔有机骨架整体材料。
2.根据权利要求1所述的一种衣康酸功能化亲水毛细管电色谱整体柱，其特征在于：所述的固定相由以下质量百分比的组分制备：1.0％-7.0％的衣康酸，4.0％-21.0％的2-丙烯酸-2-羟基-1,3-丙二酯，40.0％-53.0％的PEG 400和24.0％-42.0％的H2O，还包括催化剂过硫酸铵，过硫酸铵的用量是衣康酸和2-丙烯酸-2-羟基-1,3-丙二酯总质量的1.0％-2.5％。
3.一种衣康酸功能化亲水毛细管电色谱整体柱的制备方法，其特征在于，包括以下步骤：
(1)对石英毛细管预处理，使毛细管内壁裸漏出硅羟基；
(2)对预处理后的毛细管内壁乙烯基化修饰；
(3)配制聚合物溶液，超声混合均匀且除去气泡，所述聚合物溶液包括以下质量百分比的组分：1.0％-7.0％的衣康酸，4.0％-21.0％的2-丙烯酸-2-羟基-1,3-丙二酯，40.0％-53.0％的PEG 400，24.0％-42.0％的水，还包括催化剂过硫酸铵，过硫酸铵的用量为衣康酸和2-丙烯酸-2-羟基-1,3-丙二酯总质量的1.0％-2.5％；
(4)将步骤(3)配制的均一聚合物溶液注入到步骤(2)内壁经过乙烯基化修饰的毛细管中，两端密封，55-80℃水浴反应8-20h；反应完成后，使用甲醇冲洗毛细管整体柱，除去未反应的原料，得到衣康酸功能化亲水毛细管电色谱整体柱。
4.根据权利要求3所述的制备方法，其特征在于，步骤(1)中，采用碱溶液、酸溶液依次冲洗毛细管，使毛细管内壁裸漏出硅羟基；随后用H2O、甲醇依次冲洗毛细管；最后用氮气吹干。
5.根据权利要求4所述的制备方法，其特征在于，所述碱溶液为NaOH溶液，酸溶液为HCl溶液。
6.根据权利要求3所述的制备方法，其特征在于，步骤(1)为：石英毛细管依次使用1-1.5M NaOH溶液冲洗1-2h，1-1.5M HCl溶液冲洗1-2h，超纯水冲洗1h，甲醇冲洗20min，最后用氮气吹干。
7.根据权利要求3所述的制备方法，其特征在于，步骤(2)中，在预处理后的毛细管中注入甲基丙烯酰氧基丙基三甲氧硅烷甲醇溶液，使用Teflon两通套管连接毛细管两端成环状，放置于40-50℃水浴锅中反应10-16h；用甲醇冲洗毛细管，除去未与毛细管内壁键合的硅烷化试剂，再用氮气吹干毛细管。
8.根据权利要求7所述的制备方法，其特征在于，所述甲基丙烯酰氧基丙基三甲氧硅烷甲醇溶液，其体积分数为40-50％。
9.权利要求1或2所述的衣康酸功能化亲水毛细管电色谱整体柱在极性小分子药物分离分析中应用。
10.根据权利要求9所述的应用，其特征在于，所述的极性小分子药物为核苷类药物。
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