螺旋槽圆盘柱逆流色谱分离系统的研制及其应用评价
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摘要
目前常用的J型高速逆流色谱仪(即螺旋管行星式离心分离仪)采用的是多层盘绕的聚四氟乙烯螺旋管分离柱。多种两相溶剂体系都能够在该仪器上实现有效保留,并用于样品的分离。特别是在采用有机相/水相溶剂体系分离天然产物中的小分子化合物方面,得到成功的应用。然而,该J型高速逆流色谱仪对强极性溶剂体系和高粘度、低界面张力的双水相溶剂体系的保留率极低,限制了逆流色谱在强极性物质和生物大分子分离纯化方面的应用。高速逆流色谱仪依靠螺旋管色谱柱在做行星式运动过程中产生的阿基米德旋力实现固定相的保留,可以通过扩大螺旋管的螺距来提高固定相的保留率。在传统的J型高速逆流色谱仪中,螺距的增加受到螺旋管外径的限制。近年来,一种刻有螺旋槽的圆盘被应用于高速逆流色谱分离柱的设计,可使螺旋槽的螺距进一步增加。
本研究设计并制造了一种多层螺旋槽圆盘柱逆流色谱仪,通过一系列实验测评其对不同溶剂体系(包括有机相/水相体系和双水相体系)的影响,并选择了多种类实际样品,如黄酮类、酚类、多种二肽和蛋白类大分子等,进一步验证了其分离纯化效率。
该新型螺旋槽圆盘柱逆流色谱仪采用一个分离柱与一个配重体相平衡的设计,公转半径为9.7cm。螺旋槽圆盘分离柱由五个刻有单螺旋槽的圆盘和六个隔板间隔叠加而成,两面通过金属法兰和螺钉挤压相互密封。在上法兰和仪器公转轴上安装同样规格相互啮合的塑料齿轮,以实现分离柱的行星式运动。圆盘由聚一氯三氟乙烯(Kel-F)材料制成,刻蚀矩形螺旋槽,β值范围为0.24~0.78。螺旋槽首端口设于靠近圆盘中心位置,靠近圆盘边缘的尾端口通过导流孔与圆盘背面的通道连通,并与相邻的另一圆盘的流通槽的首端口相对应。在两圆盘之间的隔板上对应位置处设有导流小孔,实现不同圆盘上的螺线型流通槽之间的串联。分离柱总容积为74mL。
根据溶剂极性不同,分别选择了弱极性、中等极性、极性溶剂体系和双水相体系用于螺旋槽圆盘柱固定相保留率研究。通过改变流动相、色谱柱转向和流动相流向形成八种洗脱模式,分别测定了各溶剂体系在不同洗脱模式和不同流速下的固定相的保留率。研究结果表明,螺旋槽圆盘柱可使极性较弱的溶剂体系获得很高的固定相保留率。如氯仿-甲醇-水体系和正己烷-乙酸乙酯-甲醇-水体系,在以上相为流动相,采取由尾到头的洗脱模式,或以下相为流动相,采取由头到尾的洗脱模式可实现高流速(16mL/min)下的固定相的有效保留,这对于弱极性到中等极性小分子物质的快速高效分离非常有利。对于极性较强的正丁醇-醋酸-水体系及双水相体系(聚乙二醇1000-磷酸钾盐-水体系和聚乙二醇8000-葡聚糖-水体系),除U-O-T洗脱模式外,以下相为流动相,采取由内到外的洗脱模式,或以上相为流动相,采取由外到内的洗脱模式有利于固定相的高效保留。
在不同温度和转速下测定了各溶剂体系最佳洗脱模式下固定相保留率。结果表明,在20~40℃范围内,温度对固定相保留率的影响不明显,低于20℃则不利于双水相体系的保留。固定相保留率总是随着转速的增加而提高,除氯仿-甲醇-水体系外,其它各溶剂体系固定相保留率的提高速率较慢,必须达到600~800rpm的转速才能获得高的固定相保留率。
选取了四类实际样品对该螺旋槽圆盘柱逆流色谱仪的分离效果进行了验证实验。对苯二酚和邻苯二酚,银杏黄酮等小分子化合物都在较高流速下得到有效分离,证明该螺旋槽圆盘柱在植物和天然产物中小分子活性成分的高效快速分离制备方面具有重要意义。采用极性较高的正丁醇-醋酸-水体系,在4.0mL/min的高流速下,在短时间(22min)内成功分离了Leu-Tyr和Val-Tyr二肽样品。细胞色素C和肌红蛋白混合物、溶菌酶和肌红蛋白混合物、鸡蛋清等蛋白样品分别在12.5% PEG1000-12.5% K2HPO4-75% wate(rpH 9)溶剂体系和16% PEG 1000-12.5% K2HPO4-71.5% wate(rpH 8)体系中得到有效分离。以上结果证明螺旋槽圆盘柱也适用于蛋白质等生物大分子样品的分离纯化。同时该仪器还需继续改进,以改善粘度较高、分子量较大的聚合物双水相体系在应用过程中的传质效率和固定相保留率。
The commonly used high-speed counter-current chromatography (type-J HSCCC) instrument (also known as coil planet centrifuge, CPC) usually uses a multilayer coil as a separation column, which is made simply by winding a single piece of PTFE (polytetrafluoroethylene) tubing directly around the hold hub. The HSCCC centrifuge based on multilayer coil can produce a high efficient separation with good retention of stationary phase for a variety of two-phase solvent systems, especially for those organic solvent-composed systems widely used for the separation of small molecular compounds in natural products. However, it often fails to retain a satisfactory amount of the stationary phase for polar solvent system and highly viscous, low interfacial tension aqueous two-phase systems (ATPS) solvent systems, that are useful for the separation of extremely polar compounds and biological macromolecules. In the conventional type J high-speed CCC centrifuge, the retention of stationary phase almost entirely depends upon the Archimedean screw force produced by the planetary motion of coil, which can be improved by forming a spiral tube configuration to increase the radially acting centrifugal force gradient. However in the traditional spiral column design, the spiral pitch is limited by the outer diameter of the PTFE tubing. During recent years, a spiral disk column design was promoted by increasing the pitch of the spiral.
In this paper, a multiple spiral disk assembly was designed and manufactured in our laboratory, and the retention of a series of solvent systems including both typical organic-water system and ATPS was investigated. In addition, its preliminary application in the separation of small molecular flavones, peptides, and macromolecular proteins were demonstrated.
A type-J coil planet centrifuge with a 9.7 cm revolution radius was designed. A spiral disk assembly was mounted on one side and a counterweight on the other side to balance the planet centrifuge system. Theβvalues range from 0.24 to 0.78. The column consists of five single-spiral disks, each of them sandwiched by a pair of PTFE septum, they are held between a pair of stainless-steel flanges using a multiple set of screws around the inner and outer edges. The top flange is equipped with a plastic gear, which engages to an identical stationary gear mounted on the central shaft of the CPC machine. Each disk made of polymonochlorotrifluoroethylene (Kel-F) has a single rectangular spiral groove, which starts at the inner terminal (I) and ends at the outer terminal (O). It forms a spiral channel when sealed with a PTFE septum equipped with a transfer hole. The spiral channels in different disks are connected in series. The total capacity of the column is 74 mL.
A series of solvent systems including moderately polar and polar organic-aqueous solvent systems, and aqueous two-phase systems were investigated on this spiral disk column to evaluate its retention ability of different solvent systems in different rotation directions, different mobile phases, different flow-rates and different flow ways. The overall results suggested that the spiral disk column can produce excellent retention of stationary phase for moderately polar organic solvent composed systems, such as chloroform-methanol-water and hexane-ethyl acetate-methanol- water by eluting lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at high flow rate, and this makes it possible for fast separation of some small molecular compounds. The spiral disk column can also provide satisfactory retention for polar to aqueous two-phase systems such as 1-butanol-acetic acid-water, PEG 1000-K2HPO4-water and PEG8000- Dextran T500 -water at lower flow rate by eluting lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I), except for U-O-T mode.
The influence of temperature on the retention percentage of the stationary phase (Sf) was not obvious between 20℃and 40℃, lower temperature than 20℃was not suitable for viscous ATPSs. Sf increased with the increase of rotation speed (w). Except for chloroform-methanol-water system, 600-800rpm rotation speed was necessary to achieve higher Sf.
The preliminary separation of test samples proved that the spiral disk column can produce efficient separation for small molecular compounds such as hydroguinone and catechol, three flavones with less polar solvent systems composed of chloroform-methanol-water and hexane-ethyl acetate-methanol- water. This is of great significance for fast analysis and preparation of small bio-active metabolites from plant and natural resources. Acceptable resolution was also achieved when it was applied for the separation of dipeptides including Leu-Tyr and Val-Tyr by using 1-butanol-acetic acid-water (4:1:5, V/V/V) solvent system. The proteins including cytochrome C and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins were well separated by 12.5% PEG1000-12.5% K2HPO4-75% water (pH 9) and 16% PEG 1000-12.5% K2HPO4-71.5% water (pH 8) system respectively. This makes it possible for the separation of macromolecules, although it still needs to be modified to improve the retention and mass transfer in highly viscous ATPS.
本研究设计并制造了一种多层螺旋槽圆盘柱逆流色谱仪,通过一系列实验测评其对不同溶剂体系(包括有机相/水相体系和双水相体系)的影响,并选择了多种类实际样品,如黄酮类、酚类、多种二肽和蛋白类大分子等,进一步验证了其分离纯化效率。
该新型螺旋槽圆盘柱逆流色谱仪采用一个分离柱与一个配重体相平衡的设计,公转半径为9.7cm。螺旋槽圆盘分离柱由五个刻有单螺旋槽的圆盘和六个隔板间隔叠加而成,两面通过金属法兰和螺钉挤压相互密封。在上法兰和仪器公转轴上安装同样规格相互啮合的塑料齿轮,以实现分离柱的行星式运动。圆盘由聚一氯三氟乙烯(Kel-F)材料制成,刻蚀矩形螺旋槽,β值范围为0.24~0.78。螺旋槽首端口设于靠近圆盘中心位置,靠近圆盘边缘的尾端口通过导流孔与圆盘背面的通道连通,并与相邻的另一圆盘的流通槽的首端口相对应。在两圆盘之间的隔板上对应位置处设有导流小孔,实现不同圆盘上的螺线型流通槽之间的串联。分离柱总容积为74mL。
根据溶剂极性不同,分别选择了弱极性、中等极性、极性溶剂体系和双水相体系用于螺旋槽圆盘柱固定相保留率研究。通过改变流动相、色谱柱转向和流动相流向形成八种洗脱模式,分别测定了各溶剂体系在不同洗脱模式和不同流速下的固定相的保留率。研究结果表明,螺旋槽圆盘柱可使极性较弱的溶剂体系获得很高的固定相保留率。如氯仿-甲醇-水体系和正己烷-乙酸乙酯-甲醇-水体系,在以上相为流动相,采取由尾到头的洗脱模式,或以下相为流动相,采取由头到尾的洗脱模式可实现高流速(16mL/min)下的固定相的有效保留,这对于弱极性到中等极性小分子物质的快速高效分离非常有利。对于极性较强的正丁醇-醋酸-水体系及双水相体系(聚乙二醇1000-磷酸钾盐-水体系和聚乙二醇8000-葡聚糖-水体系),除U-O-T洗脱模式外,以下相为流动相,采取由内到外的洗脱模式,或以上相为流动相,采取由外到内的洗脱模式有利于固定相的高效保留。
在不同温度和转速下测定了各溶剂体系最佳洗脱模式下固定相保留率。结果表明,在20~40℃范围内,温度对固定相保留率的影响不明显,低于20℃则不利于双水相体系的保留。固定相保留率总是随着转速的增加而提高,除氯仿-甲醇-水体系外,其它各溶剂体系固定相保留率的提高速率较慢,必须达到600~800rpm的转速才能获得高的固定相保留率。
选取了四类实际样品对该螺旋槽圆盘柱逆流色谱仪的分离效果进行了验证实验。对苯二酚和邻苯二酚,银杏黄酮等小分子化合物都在较高流速下得到有效分离,证明该螺旋槽圆盘柱在植物和天然产物中小分子活性成分的高效快速分离制备方面具有重要意义。采用极性较高的正丁醇-醋酸-水体系,在4.0mL/min的高流速下,在短时间(22min)内成功分离了Leu-Tyr和Val-Tyr二肽样品。细胞色素C和肌红蛋白混合物、溶菌酶和肌红蛋白混合物、鸡蛋清等蛋白样品分别在12.5% PEG1000-12.5% K2HPO4-75% wate(rpH 9)溶剂体系和16% PEG 1000-12.5% K2HPO4-71.5% wate(rpH 8)体系中得到有效分离。以上结果证明螺旋槽圆盘柱也适用于蛋白质等生物大分子样品的分离纯化。同时该仪器还需继续改进,以改善粘度较高、分子量较大的聚合物双水相体系在应用过程中的传质效率和固定相保留率。
The commonly used high-speed counter-current chromatography (type-J HSCCC) instrument (also known as coil planet centrifuge, CPC) usually uses a multilayer coil as a separation column, which is made simply by winding a single piece of PTFE (polytetrafluoroethylene) tubing directly around the hold hub. The HSCCC centrifuge based on multilayer coil can produce a high efficient separation with good retention of stationary phase for a variety of two-phase solvent systems, especially for those organic solvent-composed systems widely used for the separation of small molecular compounds in natural products. However, it often fails to retain a satisfactory amount of the stationary phase for polar solvent system and highly viscous, low interfacial tension aqueous two-phase systems (ATPS) solvent systems, that are useful for the separation of extremely polar compounds and biological macromolecules. In the conventional type J high-speed CCC centrifuge, the retention of stationary phase almost entirely depends upon the Archimedean screw force produced by the planetary motion of coil, which can be improved by forming a spiral tube configuration to increase the radially acting centrifugal force gradient. However in the traditional spiral column design, the spiral pitch is limited by the outer diameter of the PTFE tubing. During recent years, a spiral disk column design was promoted by increasing the pitch of the spiral.
In this paper, a multiple spiral disk assembly was designed and manufactured in our laboratory, and the retention of a series of solvent systems including both typical organic-water system and ATPS was investigated. In addition, its preliminary application in the separation of small molecular flavones, peptides, and macromolecular proteins were demonstrated.
A type-J coil planet centrifuge with a 9.7 cm revolution radius was designed. A spiral disk assembly was mounted on one side and a counterweight on the other side to balance the planet centrifuge system. Theβvalues range from 0.24 to 0.78. The column consists of five single-spiral disks, each of them sandwiched by a pair of PTFE septum, they are held between a pair of stainless-steel flanges using a multiple set of screws around the inner and outer edges. The top flange is equipped with a plastic gear, which engages to an identical stationary gear mounted on the central shaft of the CPC machine. Each disk made of polymonochlorotrifluoroethylene (Kel-F) has a single rectangular spiral groove, which starts at the inner terminal (I) and ends at the outer terminal (O). It forms a spiral channel when sealed with a PTFE septum equipped with a transfer hole. The spiral channels in different disks are connected in series. The total capacity of the column is 74 mL.
A series of solvent systems including moderately polar and polar organic-aqueous solvent systems, and aqueous two-phase systems were investigated on this spiral disk column to evaluate its retention ability of different solvent systems in different rotation directions, different mobile phases, different flow-rates and different flow ways. The overall results suggested that the spiral disk column can produce excellent retention of stationary phase for moderately polar organic solvent composed systems, such as chloroform-methanol-water and hexane-ethyl acetate-methanol- water by eluting lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at high flow rate, and this makes it possible for fast separation of some small molecular compounds. The spiral disk column can also provide satisfactory retention for polar to aqueous two-phase systems such as 1-butanol-acetic acid-water, PEG 1000-K2HPO4-water and PEG8000- Dextran T500 -water at lower flow rate by eluting lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I), except for U-O-T mode.
The influence of temperature on the retention percentage of the stationary phase (Sf) was not obvious between 20℃and 40℃, lower temperature than 20℃was not suitable for viscous ATPSs. Sf increased with the increase of rotation speed (w). Except for chloroform-methanol-water system, 600-800rpm rotation speed was necessary to achieve higher Sf.
The preliminary separation of test samples proved that the spiral disk column can produce efficient separation for small molecular compounds such as hydroguinone and catechol, three flavones with less polar solvent systems composed of chloroform-methanol-water and hexane-ethyl acetate-methanol- water. This is of great significance for fast analysis and preparation of small bio-active metabolites from plant and natural resources. Acceptable resolution was also achieved when it was applied for the separation of dipeptides including Leu-Tyr and Val-Tyr by using 1-butanol-acetic acid-water (4:1:5, V/V/V) solvent system. The proteins including cytochrome C and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins were well separated by 12.5% PEG1000-12.5% K2HPO4-75% water (pH 9) and 16% PEG 1000-12.5% K2HPO4-71.5% water (pH 8) system respectively. This makes it possible for the separation of macromolecules, although it still needs to be modified to improve the retention and mass transfer in highly viscous ATPS.
引文
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[31]佘佳红,柳正良,蔡定国.高速逆流色谱分离制备白果内酯[J].中国新药杂志, 2000, 9(6) : 392-394.
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[38] Oka H, Ito Y. Separation of lac dye components using high-speed countercurrent chromatography[J]. American Laboratory, 2000, 32(8) : 24, 26-29.
[39]夏明,杜琪珍.高速逆流色谱提取分离红曲色素的研究[J].食品工业科技, 2002, 18(4) : 5-6, 17.
[40] Harada K, Ikai Y, Yamazaki Y, et al. Isolation of bacitracins A and F by high-speed counter-current chromatography[J]. Journal of chromatography, 1991, 538(1) : 203-212.
[41] Harada K, Suzuki M, Kato A, et al. Separation of WAP28294A components, a novel anti-methicillin- resistant Staphylococcus aureus antibiotic, using high-speed counter-current chromatography[J]. Journal of Chromatography A, 2001, 932(1-2) : 75-81.
[42]方东升,谢阳,陈勇,等.高速逆流色谱法分离纯化环孢菌素[J].中国抗生素杂志, 2005, 30(1) : 48-51.
[43] Shibusawa Y, Ito Y. Protein separation with aqueous-aqueous polymer systems by two types of counter-current chromatographs[J]. Journal of Chromatography, 1991, 550(1-2) : 695-704.
[44] Shibusawa Y, Hosojima T, Nakata M, et al. One-step purification of cholinesterase from human serum by CCC [J]. Journal of Liquid Chromatography and Related Technologies, 2001, 24(11-12) : 1733-1744.
[45] Shibusawa Y, Isozaki M, Yanagida A, et al. Purification of glucosyltransferase fromstreptococcus sobrinus cell culture medium by combined use of batch extraction and countercurrent chromatography with a polymer phase system[J]. Journal of Liquid Chromatography and Related Technologies, 2004, 27(14) : 2217-2234.
[46] Shibusawa Y, Misu N, Shindo H, et al. Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography[J]. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2002, 776(2) : 183-189.
[47] Yanagida A, Isozaki M, Shibusawa Y, et al. Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer twophase system[J]. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2004, 805(1) : 155-160.
[48]郅文波,邓秋云,宋江楠,等.高速逆流双水相色谱法纯化卵白蛋白[J].生物工程学报, 2005, 21(1) : 129-134.
[49] Degenhardt A, Schwarz M, Winterhalter P, et al. Evaluation of different tubing grometries for high-speed countercurrent chromatography[J]. Journal of Chromatography A, 2001, 922(1-2) : 355-358..
[50] Ito Y. Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography: I. Hydrodynamic distribution of two solvent phases in a helical column subjected to two types of synchronous planetary motion[J]. Journal of chromatography, 1984, 301 : 377-386.
[51] Ito Y. Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography: II. Phase distribution diagrams for helical and spiral columns[J]. Journal of chromatography, 1984, 301 : 387-403.
[52]饶畅,刘欣,张佩玲,等.高速逆流色谱在天然产物分离中的应用——紫杉烷类二萜及二萜生物碱的制备分离[J].药学学报, 1991 26(7) : 510-514.
[53]杨子银,屠幼英,赵勤,等.高速逆流色谱分离茶黄素单体的初步研究[J].食品科学, 2005, 26(10) : 87-90.
[54]黄健光,李总成.利用高速逆流色谱分离大黄活性成分[J].顺德职业技术学院学报, 2006, 4(1) : 31-33.
[55]王凤美,陈军辉,李磊,等.高速逆流色谱法分离制备丹酚酸B[J].天然产物研究与开发, 2006,18(1) : 100-104.
[56]蔡定国,缪平,顾明娟.高速逆流色谱法从银杏叶分离异鼠李素、山奈酚和槲皮素对照品[J].中药新药与临床药理, 1999, 10(1) : 44, 63.
[57]黄健光,李总成.利用高速逆流色谱分离邻苯二酚和对苯二酚[J].化学工业与工程, 2005,22(3) : 249-250.
[58] Awade A C, Moreau S, Molle D, et al. Two-step chromatographic procedure for the purification of hen egg white ovomucin, lysozyme, ovotransferrin and ovalbumin and characterization of purified proteins[J]. Journal of chromatography A, 1994, 677(2) : 279-288.
[59] Bergquist D H. Eggs, in Kirk Othmer Encyclopedia of Chemical Technology 4th edition[M]. New York: Wiley-Interscience, 1993.
[60] Shibusawa Y, Iino S, Shindo H, et al. Separation of chicken egg white proteins by high-speed countercurrent chromatography[J]. Journal of Liquid Chromatography and Related Technologies, 2001, 24(13) : 2007-2016.