CN116159341A - 一种三维多孔木材基整体柱及其制备方法 - Google Patents
一种三维多孔木材基整体柱及其制备方法 Download PDFInfo
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- CN116159341A CN116159341A CN202211564064.5A CN202211564064A CN116159341A CN 116159341 A CN116159341 A CN 116159341A CN 202211564064 A CN202211564064 A CN 202211564064A CN 116159341 A CN116159341 A CN 116159341A
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Images
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- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
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Abstract
本发明公开了一种以木材为骨架的三维多孔木材基整体柱及其制备方法。通过对木材裁切得到所需的形状如圆柱、圆盘形,然后将其直接修饰,或经过脱木素处理再修饰改性、偶联功能配体,获得具有蛋白质分离纯化功能的木材基整体色谱柱。可通过配体的改变,得到离子交换色谱、亲和色谱、疏水色谱、混合模式色谱等多种模式的整体色谱柱。木材独特的三维多孔结构具有优良的通透性,适用于生物大分子、病毒、细菌和颗粒的分离。木基整体柱是一种绿色、稳定、广泛应用、可实现快速分离的色谱柱。
Description
技术领域
本发明属于分离分析技术领域,具体涉及一种三维多孔木材基整体柱及其制备方法。
背景技术
由于生物质所处环境的复杂性以及对产物要求稳定性的极高要求,生物产品的分离过程成本往往占据整个生产成本的70%以上。色谱因其具有分离精度高、分离条件温和、操作简单且重复性高等特点,已成为大规模医药蛋白生产过程中使用频率最高的一种分离纯化手段。整体柱(又称为整体色谱柱,英文名为Monolithic Column)是继传统固体颗粒基质后的新一代色谱固定相,其特点在于同时拥有小的扩散孔和大的贯穿孔,这可以加快流体的传质,缩短分离时间,在一定范围内,流速增加不会显著减小分辨率,尤其在生物大分子、病毒、细菌和颗粒的分离中呈现出非常好的前景。但整体柱的制备方法、致孔剂等技术原因使其存在一些局限性,例如:整体柱孔径大小难以控制;合成大尺寸整体柱时会加剧贯穿孔的不均匀性,背压过大,柱效降低;机械强度低;缺少扩散孔等问题。将木材用作天然的整体柱,独有的层级结构和多孔特性可以成功代替目前整体柱基质成为新的色谱柱固定相。
木材独特的孔道结构极有利于流体流动通过,同时吸附拦截流体中的微小颗粒,加之其绿色、质轻、韧性好、耐冲击、可再生等特点,在生物质分离纯化领域潜力巨大。木材可以作为蛋白分离基质的优势之一是其组成成分。木材的主要组成成分是纤维素、半纤维素和木素。纤维素与半纤维素皆有碳水化合物组成,木素则为芳香族化合物,因此木材内包含丰富的羟基、酚羟基、羧基等活性基团可用于改性或直接吸附。木材中的活性基团能与酸酐、多元羧酸、酰氯、异氰酸酯、环氧化合物、醛和硅烷化合物等发生反应,形成共价键。木材的另一个优势是其天然的层级结构。木材各级结构的尺寸跨越了多个数量级,从宏观的米到微观的纳米级别。在木材的孔隙结构中,尤其是针叶材,其管胞占木质部细胞容积的90%-95%,在木材中起支撑和输导作用,这些大直径的管胞形成的“天然孔道”可以作为木基整体色谱柱的“贯穿孔”。利用木材作为整体柱材料,不需要复杂的合成步骤去形成贯穿孔以制备整体柱,有效规避整体柱中致孔剂对贯穿孔的不良影响。此外,木材还有良好的力学性能和韧度,以其作为整体柱将具有优秀的力学性能和抗压能力,良好的可生物降解性、耐用性和较低的成本。
将木材裁切成所需的形状如圆柱、圆盘形,然后将其直接修饰,或经过脱木素处理除去木材表面的杂质、木质素和其他显色基团,在维持其结构稳定性的基础上对其进行修饰、偶联不同的配体,可设计离子交换色谱、亲和色谱、疏水色谱、混合模式色谱等多种模式的整体色谱柱。经功能化处理制备的新型三维多孔木基整体色谱柱可以纯化生物物质、去污染物等,无疑对生态保护、资源回收与再利用有着重大而积极的意义。
发明内容
本发明的目的在于提供一种三维多孔木基整体色谱柱的制备方法。更详尽而言,本发明以木材为固定相,通过对其修饰改性得到一种可以应用于亲和色谱、离子交换色谱、疏水色谱、正相色谱、反相色谱、混合模式、手性拆分色谱等模式的木材基整体柱。
本发明的技术方案如下:
本发明提供三维多孔木基整体柱的制备方法,该制备方法的具体合成步骤如下:以木材为固定相,采用化学方法对其孔道表面进行修饰,让其表面连接功能性配体(也可称为配基),然后置于空色谱柱中,用于物质的分离。
本发明的三维多孔木基整体柱的制备特征结构为
[W]-(A)-(B)
其中:
[W]为木材;
(A)为中间臂,用于连接木材与功能性配体;
(B)为功能性配体,其可与目标物质通过特异性结合力或非特异性结合力相互作用。
其中(A)为非必要性结构,整体柱也可以以[W](B)结构呈现。
本发明包括色谱柱管套和固定相,固定相为天然木材或脱木素处理后木材。
本发明所述木材包括针叶材或阔叶材,优选针叶材。
本发明对木材脱木素处理采用次氯酸钠法、亚氯酸盐法、亚硫酸盐法、木素原位修饰改性法或酶处理法。
本发明中脱木素木材的脱木素程度优选为木材还能保持其原有的三维多孔结构。
本发明所述的三维多孔木材基整体柱及其制备方法,中间臂与天然木材或脱木素木材内孔道表面的羟基或羧基通过化学反应结合,结合方法包括且不限于表面接枝聚合(grafting-from)、小分子化合物或大分子聚合物直接修饰(grafting-to)、单体共聚接枝(grafting-through)等方法将中间臂与木材结合,修饰木材内部,改善木材缺点或强化优势。
本发明提供的三维多孔木材基整体柱制备方法中,所述中间臂,包括且不限于硅烷偶联剂、戊二醛、环氧氯丙烷、葡聚糖、琼脂糖、纤维素、聚乙二醇、功能性单体等。
本发明中硅烷偶联剂包括且不限于乙烯基三乙氧基硅烷(A-151)、γ-氨丙基三甲氧基硅烷(KH540)、γ-氨丙基三乙基氧基硅烷(KH550)、3-缩水甘油醚氧基丙基三甲氧基硅烷(KH560)、γ-甲基丙烯酰氧基丙基三甲氧基硅烷(KH570)、N-β-氨乙基-γ-氨丙基甲基二甲氧基硅烷(KH602)。
本发明中聚乙二醇包括双臂聚乙二醇、四臂聚乙二醇、八臂聚乙二醇、分枝聚乙二醇,其分子量为120~100000KD。
本发明中功能性单体指不饱和的、环状的或含有两个或多个官能团的低分子化合物,包括且不限于苯乙烯、甲基苯乙烯、丙烯酰胺、甲基丙烯酰胺、N-异丙基丙烯酰胺、甲基丙烯酸缩水甘油酯、甲基丙烯酸丁酯、N-烯丙基二甲基胺。
本发明中功能性配体包括且不限于亲和配体、疏水性配体、离子交换型、手性拆分配体、混合模式配体,例如蛋白A、汽巴蓝、二乙氨基乙基氯盐酸盐、甲基丙烯酸3-磺酸丙酯钾盐、金属Ni离子、正丁醛、万古霉素、Oligo(dT)、4-巯基乙基吡啶、苯丙胺、N-己胺和N-苯基-N-甲乙醇胺。
所述木材基整体柱的形状包括且不限于圆柱、圆盘、长方形、正方形、圆锥形,优选圆柱或圆盘形,木材基整体柱为一个整体的木材或多个木材基块叠加构成。
所述木材基整体柱可以用作亲和色谱、离子交换色谱、疏水色谱、正相色谱、反相色谱、混合模式色谱、手性拆分色谱。
所述木材基整体色谱柱,设计整体柱时,流动相流动方向可与针叶材木材中管胞(或阔叶材导管)方向一致或垂直。当用作生物大分子分离时,流动相流动方向优选与木材中管胞方向一致。
附图说明
图1为木基整体分离柱的合成过程示意图;
图2为一种聚合木基整体柱的扫描电镜图;
图3为KH560作为中间臂修饰脱木素木片的二乙氨基阴离子柱反应流程图;
图4为实施例2中DEAE木基整体分离柱的BSA吸附等温曲线。
具体实施方式
下面给出的实例对本发明进行具体描述,但不限制本发明,本发明的范围由权利要求限定。
实施例1:
将樟子松木材裁切成直径为2cm,高3mm的木片;将24mL功能单体甲基丙烯酸缩水甘油酯(GMA)、16mL交联剂乙二醇二甲基丙烯酸酯(EDMA)、54mL环己醇、6mL十二醇,1g过氧化苯甲酰(BPO)混合,室温下超声处理30min,再通氮气15min,除去溶液中的溶解氧,4℃放置过夜;将木片浸泡于混和溶液中4℃充分静置过夜,再通氮气15min,密封后于55℃的水浴锅中进行热引发聚24h,反应结束后,用大量的甲醇和去离子水清洗木片然后干燥;将上述木片浸入50mL 1mM甲基丙烯酸3-磺酸丙酯钾盐溶液(0.5MH2SO4将溶液pH调至0.5)中,30℃搅拌反应20h后加入5mL 1mg/mL硝酸铈铵水溶液,45℃充分振荡反应15h,反应结束后去离子水反复清洗然后冷冻干燥,得到强阳离子木基整体分离柱。
实施例2:
将新西兰辐射松木材裁切成直径为2cm,高1cm的圆柱;将裁切好的木材浸泡于7%次氯酸钠溶液中,室温搅拌5h,将木材用去离子水反复清洗后冷冻干燥;脱木素后的木材浸泡于6% KH560溶液(v/v,95%乙醇溶剂)中室温搅拌过夜,然后用去离子水清洗未结合的KH560,再次冷冻干燥;修饰后的木材放置于0.5M Na2CO3+1M NaCl,pH 11.5溶液中5h,去离子水清洗后再放置于50g/L 2-二乙氨基氯乙烷盐酸盐(DEAE-Cl)中,60℃反应15h,去离子水清洗未反应的配体然后冷冻干燥,得到DEAE弱阴离子木基色谱柱。
实施例3:
将云杉木材裁切成直径为2cm,高5cm的圆柱;将裁切好的木材浸泡于14%次氯酸钠溶液中,室温搅拌3h,将木材用去离子水反复清洗,后冷冻干燥;脱木素后的木材浸泡于6% KH560溶液(v/v,95%乙醇溶剂)中室温搅拌过夜,然后用去离子水清洗未结合的KH560,再次冷冻干燥;修饰后的木材放置于50mL 10mg/mL的汽巴蓝溶液中,加入4gNaCl混合45℃,170rpm水浴振荡反应3h使色素充分进入木材内部,向混合物中加入5mL 1M NaOH溶液继续振荡20h使反应充分进行,反应结束后用去离子水清洗未反应的配体然后冷冻干燥,得到亲和色素木基整体分离柱。
实施例4:
将云杉木材裁切成直径为2cm,高3mm的圆片;将裁切好的木材浸泡于5mL 1MNaOH溶液、2.5mL环氧氯丙烷和5mL二甲基亚砜的混合溶液中,25℃下以170rpm在恒温水浴振荡器中活化3h,用去离子水反复冲洗以除去残留的环氧氯丙烷,从而获得活化的木片;将活化后的木片浸泡于2mg/mL的蛋白A甲醇溶液中,40℃下于水浴中振摇16h,反应结束后用体积分数为80%的甲醇反复洗涤;然后将木柱转移至1M乙醇胺(pH值为8)中,于50℃过夜;最后,用1M Tris-HCl+0.5M NaCl缓冲液(pH值为8)彻底冲洗木柱3次后冷冻干燥,得到亲和木基整体分离柱。
实施例5:
将辐射松木材裁切成直径为2cm,高5cm的圆柱;将裁切好的木材浸泡于14%次氯酸钠溶液中,室温搅拌3h,将木材用去离子水反复清洗后冷冻干燥;将脱木素木材浸泡于100mL去离子水中,70℃加热搅拌4h,再加入10%戊二醛水溶液,继续加热搅拌1h后去离子水反复清洗后冷冻干燥;戊二醛修饰后的木材浸泡于50mL正丁醛(pH 6.0)中,室温搅拌10h后,去离子水和无水乙醇反复洗涤,冷冻干燥,得到木基整体疏水分离柱。
实施例6:
将辐射松木材裁切成直径为2cm,高3mm的圆片;将裁切好的木材浸泡于5mL1MNaOH溶液、2.5mL环氧氯丙烷和5mL二甲基亚砜的混合溶液中,25℃下以170rpm在恒温水浴振荡器中活化3h,用去离子水反复冲洗以除去残留的环氧氯丙烷,从而获得活化的木片;将活化后的木材置于20mL 300mg/mL的葡聚糖T40溶液中,并在恒温水浴振荡器中以170rpm震荡1h以使Dextran T40均匀扩散到木材孔内;随后,再加入6mL 0.8MNaOH、40mg硼氢化钠,并30℃反应15h,反应结束后用去离子水反复清洗;1g的均苯三甲酸置于10mL氯化亚砜中,60℃反应6h后旋蒸除去溶剂,再加入10ml无水N,N-二甲基甲酰胺和1.2g万古霉素于80℃下反应12h,反应结束后将所得产物旋蒸除去N,N-二甲基甲酰胺,得到羧酸化的万古霉素;将以上实验得到的羧酸化的万古霉素和修饰后的木材置于二甲基亚砜溶剂中,室温搅拌48h,反应结束后,去离子水反复清洗,得到负载万古霉素的木基整体色谱柱。
实施例7:
将落叶松木材裁切成直径为2cm,高3mm的圆片;将裁切好的木材浸泡于5mL1MNaOH溶液、2.5mL环氧氯丙烷和5mL二甲基亚砜的混合溶液中,25℃下以170rpm在恒温水浴振荡器中活化3h,用去离子水反复冲洗以除去残留的环氧氯丙烷,从而获得活化的木片;活化后的木片加入50mL pH为7的亚氨基二乙酸溶液和50mL pH为9.5的MaHCO3缓冲液,37℃200rpm震荡反应24h后用去离子水反复清洗;再将木片加入50mL的0.1M NiSO4溶液中浸泡3h后,去离子水反复洗涤,冷冻干燥,得到金属亲和木基整体分离柱。
实施例8:
将云杉木材裁切成直径为2cm,高5cm的圆柱;将裁切好的木材浸泡于14%次氯酸钠溶液中,室温搅拌3h,将木材用去离子水反复清洗,后冷冻干燥;脱木素后的木材浸泡于6% KH560溶液(v/v,95%乙醇溶剂)中室温搅拌过夜,然后用去离子水清洗未结合的KH560,再次冷冻干燥;将硅烷化修饰的木片与50mL 30%氨基聚乙二醇2000水溶液混合,60℃加热反应10h,反应结束后去离子水反复清洗多次;再将反应后的木片与30mL 10mg/mL的Oligo(dT)18溶液在pH 8的条件下,37℃,200rpm振荡孵育24h,反应后去离子水反复洗涤,冷冻干燥,得到Oligo(dT)木基整体分离柱。
实施例9:
将落叶松裁切成长30mm,宽30mm,厚3mm的木片;将裁切好的木材浸泡于14%次氯酸钠溶液中,室温搅拌3h,将木材用去离子水反复清洗,后冷冻干燥;脱木素后的木材浸泡于6% KH560溶液(v/v,95%乙醇溶剂)中室温搅拌过夜,然后用去离子水清洗未结合的KH560,再次冷冻干燥;放置于50mL乙二胺中,65℃反应10h,反应结束后,用大量去离子水清洗木片;将木片置于50mg/mL的肝素钠甲醇溶液孵育24h,再加入30mg硼氢化钠孵育12h,反应后去离子水反复洗涤,冷冻干燥,得到肝素亲和木基整体分离柱。
Claims (12)
1.一种三维多孔木材基整体柱及其制备方法,其特征在于,所述木材基整体柱的固定相以木材为骨架,采用化学方法对其孔道表面进行处理和修饰,让其表面连接功能性配体,然后置于空色谱柱中,用于物质的分离纯化。
2.根据权利要求1所述的三维多孔木材基整体柱,特征在于,其结构式为[W]-(A)-(B)
其中:
[W]为木材;
(A)为中间臂,用于连接木材与功能性配体;
(B)为功能性配体,其可与目标物质通过特异性结合力或非特异性结合力相互作用;
其中(A)为非必要性结构,整体柱也可以以[W](B)结构呈现。
3.根据权利要求1-2所述的三维多孔木材基整体柱及其制备方法,所述木材为针叶材或阔叶材,优选针叶材,包括天然木材或脱木素处理的木材。
4.根据权利要求1-3中所述的三维多孔木材基整体柱及其制备方法,脱木素木材制备方法包括且不限于次氯酸盐法、亚氯酸盐法、亚硫酸盐法、木素原位修饰改性法、酶处理法;
脱木素木材的脱木素程度优选为木材能保持其原有的三维多孔结构。
5.根据权利要求1-4中所述的三维多孔木材基整体柱及其制备方法,中间臂与天然木材或脱木素木材内孔道表面的羟基或羧基通过化学反应结合,结合方法包括且不限于小分子化合物或大分子聚合物直接修饰(grafting-to)、表面接枝聚合(grafting-from)、单体共聚接枝(grafting-through)。
6.根据权利要求5中所述的三维多孔木材基整体柱及其制备方法,所述中间臂,包括且不限于硅烷偶联剂、戊二醛、环氧氯丙烷、葡聚糖、琼脂糖、纤维素、聚乙二醇、功能性单体。
7.根据权利要求6中所述的三维多孔木材基整体柱及其制备方法,硅烷偶联剂包括且不限于乙烯基三乙氧基硅烷(A-151)、γ-氨丙基三甲氧基硅烷(KH540)、γ-氨丙基三乙基氧基硅烷(KH550)、3-缩水甘油醚氧基丙基三甲氧基硅烷(KH560)、γ-甲基丙烯酰氧基丙基三甲氧基硅烷(KH570)、N-β-氨乙基-γ-氨丙基甲基二甲氧基硅烷(KH602)。
8.根据权利要求6中所述的三维多孔木材基整体柱及其制备方法,所述聚乙二醇包括双臂聚乙二醇、四臂聚乙二醇、八臂聚乙二醇、分枝聚乙二醇,其分子量为120~100000KD。
9.根据权利要求6中所述的三维多孔木材基整体柱及其制备方法,所述功能性单体指不饱和的、环状的或含有两个或多个官能团的低分子化合物,包括且不限于苯乙烯、甲基苯乙烯、丙烯酰胺、甲基丙烯酰胺、N-异丙基丙烯酰胺、甲基丙烯酸缩水甘油酯、甲基丙烯酸丁酯、N-烯丙基二甲基胺。
10.根据权利要求1-9中所述的三维多孔木材基整体柱及其制备方法,所述功能性配体包括且不限于亲和配体、疏水性配体、离子交换型配体、手性拆分配体、混合模式配体,例如蛋白A、肝素、汽巴蓝、二乙氨基乙基化合物、甲基丙烯酸3-磺酸丙酯钾盐、金属Ni离子、正丁醛、万古霉素、Oligo(dT)、4-巯基乙基吡啶、苯丙胺、N-己胺和N-苯基-N-甲乙醇胺。
11.根据权利要求1-10中所述的三维多孔木材基整体柱及其制备方法,所述木材基整体柱的形状包括且不限于圆柱、圆盘、长方形、正方形、圆锥形,优选圆柱形或圆盘形,木材基整体柱为一个整体的木材或多个木材基块叠加构成。
12.根据权利要求1-11中所述的三维多孔木材基整体柱及其制备方法,所述木材基整体柱可以用作亲和色谱、离子交换色谱、疏水色谱、正相色谱、反相色谱、混合模式色谱、手性拆分色谱。
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